Method of assay having calibration within the assay

ABSTRACT

A method of assay for a ligand in a sample is described in which calibration occurs within the assay. This is achieved utilizing a measurement region and one ore more calibration regions. In at least one of the calibration regions a non-zero signal results, either because of the presence of a calibration reagent or as a result of a binding reaction analogous to that which takes place in the measurement region.

This is a continuation of application Ser. No. 08/064,107, filed on May24, 1993, and now abandoned.

This invention relates to a method of assay of chemical, biochemical orbiological entities, to devices for use in such a method, to a method ofmanufacture of such devices and to the use of such devices.

There is now a great interest in the development of assay techniques forthe detection and measurement of the presence of an analyte in a sample,and the various methods available have been extensively reviewed, forexample in Biosensors: Fundamentals and Applications, edited by A. P. F.Turner, I. Karube, G. S. Wilson, Oxford Scientific Publications, 1987.Current techniques, however, are highly sensitive no temperature,reagent stability, incubation and development time, and other conditionsand interfering factors which may affect the level of the signalobserved. Accordingly, the precision of known assay techniques islimited by the method of calibration, which usually involves carryingout an assay on a standard sample. For example, for assays which involvean antibody, the immunological binding reactions which occur arefrequently irreversible. Thus any calibration steps need to be carriedout using a separate device or devices (preferably from the samemanufacturing batch), which inevitably introduces errors.

The need for a separate calibration step involving the use of additionalsensing devices can be avoided by using in the assay a device which isprovided with appropriate reagents disposed in separate zones wherebythe calibration step is effected within the assay procedure. The use ofan assay method wherein a separate calibration step is effected withinthe assay procedure serves two main purposes, namely i) to confirm thatthe various reagents used in the assay procedure are performingaccording to their specification, and ii) to define a certainconcentration level within the sample on test, and thereby to compensatefor background interference (e.g. background fluorescence), temperatureand pH changes and other factors originating from the sample matrixwhich may alter the level of the observed signals.

EP-A-0093613 (SYVA) discloses an assay method for determining thepresence of an analyte in a sample by means of a measurement region anda calibration region. The method involves the use of a common species inboth of the regions which gives a signal at the measurement regionrelated to the amount of analyte in the sample, and a signal at thecalibration region independent of the analyte concentration. The commonspecies is captured in the calibration region by means of a differentbinding reaction to that which takes place in the measurement region.

The assay method disclosed in EP 0093613 therefore provides for aseparate calibration within the assay and to a certain extent does servepurpose ii) above. However such a method of calibration suffers from anumber of disadvantages. The use of a different binding reaction in thecalibration region means that the behaviour of the two binding reactions(i.e. in the measurement and calibration regions respectively) will notbe the same in terms of various factors e.g. susceptibility to pH andtemperature, reagent stability and reagent aging. The binding reactionin the calibration region will also be affected differently by thesample matrix and so no compensation can be made for changes occurringto the signal arising from the binding reaction in the measurementregion as a result of the sample matrix. There will also be no check onthe performance of the binding reaction occurring in the measurementregion (c.f. purpose i) above). Furthermore, the manufacture of devicesfor such an assay is made more complex by needing two different sets ofreagents.

We have now developed an alternative assay method which overcomes thesedisadvantages of the method of EP 0093613 and which still fulfills thepurposes i) and ii) above.

Thus, according to one aspect of the present invention we provide amethod of assay for a ligand in a sample which method comprises thesteps of:

i) incubating the sample, if desired together with one or more ancillaryreagents, in contact with a surface ("the measurement surface") whichsurface carries an immobilised reagent ("the measurement reagent")appropriate to the assay technique employed whereby if ligand is presentin the sample a complex involving said measurement reagent and saidligand and/or (if present) said ancillary reagent(s) is formed givingrise to a detectable signal which is a first function of the amount ofligand (if any) present in the sample;

ii) simultaneously or sequentially contacting the sample, if desiredtogether with one or more ancillary reagents, with a further surface("the calibration surface") onto which is immobilised a reagent ("thecalibration reagent") appropriate to the assay technique employed, thecalibration reagent either being such as to give rise to a non-zerosignal or being such as to form a complex involving said ligand and/orsaid ancillary reagent(s) whereby any such complex gives rise to anon-zero signal and is formed as a result of the interaction of bindingsites identical in structure to those involved in the formation of theaforesaid complex formed on the measurement surface (or, where no suchcomplex is formed, which would be formed if ligand were present) eitherbetween the measurement reagent and the ligand or, where the ligand isnot involved in said complex, between the measurement reagent and saidancillary reagent(s), the signal being either a second function of orindependent of the amount of ligand (if any present in the sample;

iii) optionally simultaneously or sequentially contacting the sample, ifdesired together with one or more ancillary reagents, with a furthercalibration surface ("the auxiliary calibration surface") onto which isimmobilised a reagent ("the auxiliary calibration reagent"), theauxiliary calibration reagent being such as to give rise to a signal(zero or non-zero as herein defined) which is either a third function ofor independent of the amount of ligand (if any) present in the sample;and

iv) monitoring the signals arising from the measurement surface, fromthe calibration surface and, when present, from the auxiliarycalibration surface by a method appropriate to the assay techniqueemployed and, by comparing the signals arising from the aforesaidsurfaces, thereby determining (using an appropriate algorithm tocalibrate the signal arising from the measurement surface) whetherand/or the extent to which the ligand under assay is present in thesample.

In the embodiments described hereinafter wherein there occurs at thecalibration surface a binding reaction analogous to that which occurs atthe measurement surface (if ligand is present in the sample) purpose i)indicated above is achieved i.e. there may be confirmation that thereagents in the complex which give rise to the signal have not degradedor that the binding reactions are occurring satisfactorily i.e. thebinding partners in such reactions have not degraded. Purpose ii) mayalso be achieved in these embodiments.

In the embodiments described hereinafter wherein at the calibrationsurface the calibration reagent gives rise to the desired non-zerosignal without there being a binding reaction to any ancillaryreagent(s), purpose ii) indicated above is achieved.

The use of an optional calibration surface supplements the calibrationachieved by the calibration surface. The auxiliary calibration surfacemay utilize similar reagents to those used in the calibration surface ormay utilise reagents wherein binding reactions occur at the auxiliarycalibration surface distinct from those which occur at either themeasurement surface or the calibration surface, either to give a furthernon-zero signal or a zero-signal as defined herein.

According to a further aspect of the present invention there is provideda biosensor device suitable for use in assaying a ligand in a sample bya method of assay as hereinbefore defined, said device comprising ameasurement surface carrying a measurement reagent and a calibrationsurface carrying a calibration reagent optionally together with one ormore auxiliary calibration surfaces each carrying an auxiliarycalibration reagent, said measurement reagent, calibration reagent andauxiliary calibration reagent each being as defined above.

In step ii) above, where the signal is a second function of the amountof ligand present in the sample, this second function is different tothe first function specified in step i). In step iii) above, where thesignal is a third function of the amount of ligand present in thesample, this third function is different to the first function specifiedin step i) and may be the same as, but is preferably different from, thesecond function specified in step ii).

Where an auxiliary calibration surface is present, the calibrationreagent and auxiliary calibration reagent will be chosen such that thesignals arising from the calibration surface and from the auxiliarycalibration surface are not identical. Such non-identical signals canarise where the signal arising from the calibration surface and thesignal arising from the auxiliary calibration surface is the samefunction of the amount of ligand present in the sample. One example iswhere the calibration reagent and auxiliary calibration reagent are thesame but the amounts of ancillary reagent(s) which form a complex withthe calibration reagent and auxiliary calibration reagent differ.Another example is where the calibration reagent and auxiliarycalibration reagent both give rise to a signal without the need for anancillary reagent and are present in differing amounts. If it is found,despite such a choice of calibration reagent and auxiliary calibrationreagent, that identical signals arise, then device failure (e.g. due toextremes of sample pH, too high a sample background signal or reagentdegradation) is indicated and the assay can be rejected; this is afurther advantage of the present invention.

For a qualitative method of assay for a ligand in a sample, preferablyone auxiliary calibration surface is present. For a semi-quantitativemethod at least one auxiliary calibration surface is present. For aquantitative method, the number of auxiliary calibration surfacespresent is preferably greater than one, more preferably greater than orequal to four.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic section through a fluorescence capillary filldevice (hereinafter FCFD).

FIG. 2 is a planned view of the device of FIG. 1.

FIG. 3(a) is a diagrammatic illustration of reagents contained inregions T, R and S for a competition-type assay.

FIG. 3(b) is a diagrammatic illustration of reagent T in a firstembodiment of the invention for a competition-type assay.

FIG. 3(c) is a diagrammatic illustration of reagent T in a secondembodiment of the invention for a competition-type assay.

FIG. 3(d) is a diagrammatic illustration of reagent T in a thirdembodiment of the invention for a competition-type assay.

FIG. 3(e) is a diagrammatic illustration of region R in a first exampleof the invention for a competition-type assay.

FIG. 3(f) is a diagrammatic illustration of region R in a second exampleof the invention for a competition-type assay.

FIG. 3(g) is a diagrammatic illustration of region R in a third exampleof the invention for a competition-type assay.

FIG. 3(h) is a diagrammatic illustration of region R in a fourth exampleof the invention for a competition-type assay.

FIG. 3(i) is a diagrammatic illustration of region R in a fifth exampleof the invention for a competition-type assay.

FIG. 3(j) is a diagrammatic illustration of region R in a sixth exampleof the invention for a competition-type assay.

FIG. 3(k) is a diagrammatic illustration of region R in a seventhexample of the invention for a competition-type assay.

FIG. 3(l) is a diagrammatic illustration of region S in a first exampleof the invention for a competition-type assay.

FIG. 3(m) is a diagrammatic illustration of region S in a second exampleof the invention for a competition-type assay.

FIG. 3(n) is a diagrammatic illustration of region S in a third exampleof the invention for a competition-type assay.

FIG. 3(p) is a diagrammatic illustration of region S in a fourth exampleof the invention for a competition-type assay.

FIG. 3(q) is a diagrammatic illustration of region R of a firstadditional example of the invention for a competition-type assay.

FIG. 3(r) is a diagrammatic illustration of region R of a secondadditional example of the invention for a competition-type assay.

FIG. 3(s) is a diagrammatic illustration of region R of a thirdadditional example of the invention for a competition-type assay.

FIG. 3(t) is a diagrammatic illustration of region S in a firstembodiments of the invention for a competition-type

FIG. 3(u) is a diagrammatic illustration of region S in a firstembodiments of the invention for a competition-type assay.

FIG. 3(v) is a diagrammatic illustration of region R in a furtherembodiment of the invention for a competition-type assay.

FIG. 4(a) diagrammatically illustrates a first example of measurementregion T for a competition-type assay.

FIG. 4(b) diagrammatically illustrates a second example of measurementregion T for a competition-type assay.

FIG. 4(c) diagrammatically illustrates a third example of measurementregion T for a competition-type assay.

FIG. 4(d) diagrammatically illustrates a first example of a positivecalibration region R for a competition-type assay.

FIG. 4(e) diagrammatically illustrates a second example of a positivecalibration region R for a competition-type assay.

FIG. 4(f) diagrammatically illustrates a first example of a high signalcalibration region R for a competition-type assay.

FIG. 4(g) diagrammatically illustrates a second example of a high signalcalibration region R for a competition-type assay.

FIG. 4(h) diagrammatically illustrates a third example of a high signalcalibration region R for a competition-type assay.

FIG. 4(i) diagrammatically illustrates a fourth example of a high signalcalibration region R for a competition-type assay.

FIG. 4(j) diagrammatically illustrates a fifth example of a high signalcalibration region R for a competition-type assay.

FIG. 4(k) diagrammatically illustrates a sixth example of a high signalcalibration region R for a competition-type assay.

FIG. 4(l) diagrammatically illustrates a seventh example of a highsignal calibration region R for a competition-type assay.

FIG. 4(m) diagrammatically illustrates a eighth example of a high signalcalibration region R for a competition-type assay.

FIG. 4(n) diagrammatically illustrates a first example of a zero signalcalibration region S for a competition-type assay.

FIG. 4(p) diagrammatically illustrates a second example of a zero signalcalibration region S for a competition-type assay.

FIG. 4(q) diagrammatically illustrates a third example of a zero signalcalibration region S for a competition-type assay.

FIG. 4(r) diagrammatically illustrates a fourth example of a zero signalcalibration region S for a competition-type assay.

FIG. 4(s) diagrammatically illustrates region R for a firstcompetition-type assay.

FIG. 4(t) diagrammatically illustrates region R for a secondcompetition-type assay.

FIG. 5(a) diagrammatically illustrates a sandwich assay measurementregion T.

FIG. 5(b) diagrammatically illustrates a sandwich assay positivecalibration region R.

FIG. 5(c) diagrammatically illustrates high signal calibration region Rof a first sandwich assay embodiment of the invention.

FIG. 5(d) diagrammatically illustrates high signal calibration region Rof a second sandwich assay embodiment of the invention.

FIG. 5(e) diagrammatically illustrates high signal calibration region Rof a third sandwich assay embodiment of the invention.

FIG. 5(f) diagrammatically illustrates high signal calibration region Rof a fourth sandwich assay embodiment of the invention.

FIG. 5(g) diagrammatically illustrates high signal calibration region Rof a fifth sandwich assay embodiment of the invention.

FIG. 5(h) diagrammatically illustrates high signal calibration region Rof a sixth sandwich assay embodiment of the invention.

FIG. 5(i) diagrammatically illustrates zero signal calibration region Sof a first embodiment of sandwich assays.

FIG. 5(j) diagrammatically illustrates zero signal calibration region Sof a second embodiment of sandwich assays.

FIG. 6 shows schematically a simple fluorimetry apparatus.

FIG. 7 is a plot of signal versus time in zone V of example 1.

FIG. 8 is a plot of signal versus log concentration in zone IV ofexample 1.

FIG. 9 is a plot of signal versus log concentration of zones IV and V inexample 2.

FIGS. 10(a) and 10(b) are plots of positive results versus logconcentration in example 3.

FIG. 11 is a plot of signal versus log concentration of zones IV and Vin example 4.

FIG. 12 is a plot of signal versus log concentration of zones IV, V andVI in example 5.

FIG. 13 is a plot of signal versus vernier distance in example 5.

FIG. 14 is a plot of signal versus log concentration of zones IV, V andVI in example 6.

FIG. 15 is a plot of signal versus log concentration of zones IV and Vin example 6.

FIG. 16 is a plot of signal versus log concentration of zones V and VIin example 6.

FIG. 17 is a plot of signal versus log concentration of zones IV and Vin example 7.

FURTHER DESCRIPTION OF THE INVENTION

The method of assay according to the invention is applicable to a widevariety of assay techniques including direct assays, competition assaysand sandwich assays.

The term "direct assay" is used herein to mean an assay in which toancillary reagent is required and hence in which binding of sampleligand to an appropriate specific binding partner directly modulates thesignal being measured, for example certain assays using surface plasmonresonance or piezoelectric biosensors. However, such biosensorssometimes use labels to enhance their performance (for example asdescribed in EP-A-276142). The use of such indirect assay techniques asapplied to the method of the present invention is encompassed by thepresent application.

The term "zero signal" as used above denotes the background signal forthe assay concerned. The term "non-zero signal" is to be construedaccordingly.

In a direct or sandwich assay, the zero signal will be the signalobtained when to analyte is present. In a competition assay, the zerosignal will be the signal corresponding to the low asymptote of theappropriate assay curve and will therefore not be the signal obtainedwhen to analyte is present.

In direct assays, and in sandwich assays the detectable signal will ingeneral be proportional to the quantity of ligand present in the sample.In competition assays, a complex between measurement reagent and theancillary reagent will be formed whether or not ligand is present in thesample but the detectable signal will depend on the quantity ofancillary reagent complexed; this will in general be inverselyproportional to the quantity of ligand present in the sample. The term"competition assay" as used herein includes within its scope, where thecontext so permits, displacement assays, e.g. assays in which themeasurement reagent is pre-complexed with an appropriate ancillaryreagent and this pre-complex is subsequently incubated with samplewhereby at least a portion of any ligand present in the sample displacesa corresponding amount of ancillary reagent.

Thus, in one type of assay in accordance with an embodiment of thepresent invention:

in step i) the measurement reagent (or optionally an ancillary reagentprecomplexed with or capable of forming a complex involving themeasurement reagent) is a specific binding partner for the ligand underassay; and

in step ii) a ligand analogue is present as an ancillary reagent and thecalibration reagent (or optionally an ancillary reagent precomplexedwith or capable of forming a complex involving the calibration reagent)is a specific binding partner for the ligand under assay; and

in step iii) either a) the auxiliary calibration reagent and ancillaryreagent(s) are equivalent to the calibration reagent and ancillaryreagent(s) respectively defined in step ii) above or b) a liganddistinct from the ligand under assay is present as an ancillary reagentand the auxiliary calibration reagent (or optionally an ancillaryreagent precomplexed with or capable of forming a complex involving theauxiliary calibration reagent) is a specific binding partner for theligand distinct from the ligand under assay or c) the auxiliarycalibration reagent is a binding partner non-specific for the ligandunder assay.

In a competition assay according to a further embodiment of the presentinvention:

in step i) either a) a ligand analogue is present as an ancillaryreagent and the measurement reagent (or optionally an ancillary reagentprecomplexed with or capable of forming a complex involving themeasurement reagent) is a specific binding partner for the ligand underassay or b) an optionally labelled specific binding partner for theligand under assay is present as an ancillary reagent and themeasurement reagent (or optionally an ancillary reagent precomplexedwith or capable of forming a complex involving the measurement reagent)is a ligand analogue; and

in step ii) either a) a ligand analogue is present as an ancillaryreagent and the calibration reagent (or optionally an ancillary reagentprecomplexed with or capable of forming a complex involving thecalibration reagent) is a specific binding partner for the ligand underassay or b) an optionally labelled specific binding partner for theligand under assay is present as an ancillary reagent and thecalibration reagent (or optionally an ancillary reagent precomplexedwith or capable of forming a complex involving the calibration reagent)is a ligand analogue or c) the calibration reagent gives rise to thedesired non-zero signal without the need for presence of an ancillaryreagent; and

in step iii) either a) the auxiliary calibration reagent and ancillaryreagent(s) are equivalent to the calibration reagent and ancillaryreagent(s) respectively defined in step ii) above or b) an optionallylabelled ligand distinct from the ligand under assay is present as anancillary reagent and the auxiliary calibration reagent (or optionallyan ancillary reagent precomplexed with or capable of forming a complexinvolving the auxiliary calibration reagent) is a specific bindingpartner for the ligand distinct from the ligand under assay or c) theauxiliary calibration reagent is a binding partner non-specific for anyancillary reagent(s) present or d) the auxiliary calibration reagentgives rise to the desired zero signal without the need for the presenceof an ancillary reagent.

In a sandwich assay according to a still further embodiment of thepresent invention:

in step i) an optionally labelled specific binding partner for theligand under assay is present as an ancillary reagent and themeasurement reagent (or optionally an ancillary reagent precomplexedwith or capable of forming a complex involving the measurement reagent)is a further specific binding partner for the ligand under assay thesaid further specific binding partner being directed to an epitope ofthe ligand under assay different to the epitope to which the optionallylabelled specific binding partner is directed; and

in step ii) either a) the calibration reagent (or optionally anancillary reagent precomplexed with or capable of forming a complexinvolving the calibration reagent) is a specific binding partner for theligand under assay, an optionally labelled specific binding partner forthe ligand under assay is present as an ancillary reagent and a knownamount of the ligand under assay precomplexed to its optionally labelledspecific binding partner is present as a yet further ancillary reagentor b) an optionally labelled specific binding partner for the ligandunder assay is present as an ancillary reagent and the calibrationreagent (or optionally an ancillary reagent precomplexed with or capableof forming a complex involving the calibration reagent) is a knownamount of the ligand under assay precomplexed to its immobilizedspecific binding partner or c) the calibration reagent gives rise to thedesired non-zero signal without the need for presence of an ancillaryreagent; and

in seep iii) either a) the auxiliary calibration reagent and ancillaryreagent(s) are equivalent to the calibration reagent and ancillaryreagent(s) respectively defined in step ii) above or b) a liganddistinct from the ligand under assay is present as an ancillary reagentand the calibration reagent (or optionally an ancillary reagentprecomplexed with or capable of forming a complex involving thecalibration reagent) is an optionally labelled specific binding partnerfor the ligand distinct from the ligand under assay or c) the auxiliarycalibration reagent is an optionally labelled binding partnernon-specific for any ancillary reagent(s) present or d) the auxiliarycalibration reagent gives rise to the desired zero signal without theneed for the presence of an ancillary reagent.

The term "ligand analogue" as used herein denotes a species which iscapable of binding to the same epitopic site of the same specificbinding partner as the ligand under assay, and includes inter aliawithin its scope a known amount of the ligand under assay or a labelledaliquot of the said ligand.

A wide variety of devices may be used to perform the method of thepresent invention including for example dipstick or `test-strip`biosensors, devices using a `sample flow-through` configuration ordevices employing sample containment. Examples of biosensors which maybe used in the method of the present invention include sensors involvingsurface plasmon resonance, piezoelectric and total internal reflectancetechniques. A preferred device to carry out the method of the presentinvention is a capillary fill device, especially a fluorescencecapillary fill device, for example the type of device described inEP-A-171148 or in WO-90/14590. Such capillary fill devices may be usedsingly or in a suitable holder such as described in WO 90/1830.

As described in EP-A-171148, a capillary fill device (hereinafter CFD)typically consists of two plates of transparent material, e.g. glass,separated by a narrow gap or cavity. One plate acts as an opticalwaveguide and carries an immobilised reagent appropriate to the test tobe carried out in the device. As described in WO-90/14590, the othertransparent plate can carry on its surface remote from the cavity alayer of light-absorbing or opaque material. For use in a competitionassay, the immobilised reagent may for example be a specific bindingpartner to the ligand desired to be detected and one of the plates maycarry a dissoluble reagent comprising ligand analogue, labelled with afluorescent dye (the ancillary reagent). When a sample is presented toone end of the CFD, it is drawn into the gap by capillary action anddissolves the ancillary reagent. In a competition assay for an antigen,the fluorescently labelled antigen analogue will compete with sampleantigen for the limited number of antibody binding sites immobilised onthe waveguide. Because the capillary gap is narrow (typically about 100microns), the reaction will generally go to completion in a short time,possibly less than 5 minutes depending upon the sample matrix andantibody affinity. Thus for a competition assay, the amount offluorescently labelled antigen which becomes indirectly bound to thewaveguide by virtue of complex formation will be inversely proportionalto the concentration of antigen in the sample. In a sandwich assay, thewaveguide will carry a specific binding partner for the ligand desiredto be detected and either one of the plates will carry a dissolublereagent comprising a further specific binding partner labelled with afluorescent dye (the ancillary reagent). In a sandwich immunoassay foran antigen, a sample antigen will form a sandwich complex with afluorescently labelled antibody and an antibody immobilised on thewaveguide. Thus, for a sandwich immunoassay, the amount of fluorescentlylabelled antibody which becomes indirectly bound to the waveguide byvirtue of complex formation will be directly proportional to theconcentration of antigen in the sample.

The term "antigen" as used herein will be understood to include bothantigenic species (for example, proteins, bacteria, bacterial fragments,cells, cell fragments and viruses) and haptens which may be renderedantigenic under suitable conditions.

According to a preferred embodiment of the device according to theinvention, we provide a specifically-reactive sample-collecting andtesting device for use in an assay for a ligand, possessing a cavity orcavities, one surface of the or each cavity having three zones I, II andIII mutually separated and each zone carrying a layer comprising, inreleasable form, a reagent suitable for the desired assay, said surfacebeing a surface of a first solid plate fashioned of transparentmaterial, wherein the wall of the or each cavity opposite to said firstplate comprises a second plate fashioned of transparent material andadapted to act as a light-transmissive waveguide, the second platehaving on its surface adjacent the cavity three zones IV, V and VIcorresponding in orientation to the aforementioned zones I, II and IIIrespectively, each of zones IV, V and VI carrying a layer comprising animmobilised reagent suitable for the desired assay. The first plateadvantageously carries on its external face an opaque coating.

For convenience, in the more detailed description such a device, thereagents carried by the aforementioned zones I, II, III, IV, V and VIwill be designated as follows:

    ______________________________________                                        Zone          Reagent                                                         ______________________________________                                        I.      (top plate)                                                                             X        (ancillary reagent in                                                         soluble, releasable form)                          II.     (top plate)                                                                             Y        (ancillary reagent in                                                         soluble, releasable form)                          III.    (top plate)                                                                             Z        (ancillary reagent in                                                         soluble, releasable form)                          IV.     (baseplate)                                                                             A        (immobilised reagent)                              V.      (baseplate)                                                                             B        (immobilised reagent)                              VI.     (baseplate)                                                                             C        (immobilised reagent)                              ______________________________________                                    

The terms "top plate" and "baseplate" are used purely for convenience ofdescription and their use is not intended to limit in any way theconfiguration in which the device may be used.

The arrangement of the aforementioned zones is such that zone I ispaired together with zone IV, zone II is paired together with zone V andzone III is paired together with zone VI, such that one of said pairsprovides the region of the device which gives rise to a measurement ofthe amount of ligand it is desired to assay (the "measurement region")and the other two pairs provide regions of the device which give rise tomeasurements which can be used as control or calibration parameters (the"calibration regions").

CFDs for use in the method of the invention may if desired contain morethan one auxiliary calibration zone; and may if desired contain multipleassay zones enabling simultaneous or sequential assays for differentligands in the same sample to be conducted. For example, the devicecould contain a first measurement zone and a calibration zone as hereindefined for one assay together with a further measurement zone for adifferent assay. The calibration zone would also serve as a calibrationfor the further measurement zone although such calibration would differfrom that for the first measurement zone. Additionally, auxiliarycalibration zones could be included as desired.

The identities of the reagents X, Y, Z, A, B and C will depend both onthe ligand it is desired to assay and on the assay methodology. Thereagents carried in the zones on the first transparent plate may becontained within a dissoluble layer of a suitable material. Afterdeposition of the soluble reagent, a capping layer e.g. polyvinylalcohol (PVA) may be placed upon the reagent, which capping layer delaysthe dissolution of the reagent for a few seconds after the addition ofthe sample to the device. This is to prevent the reagents being washedfrom one zone to another thereby precluding an accurate assay. Thecavity or cavities of the device are preferably of a dimension smallenough to enable sample liquid to be drawn into the cavity by capillaryaction, although any other method of filling said cavities may beemployed. The zones on the first transparent plate and thereby thecorresponding zones on the second transparent plate may be arrangedeither in tandem or in any other geometrical arrangement which maintainsthe integrity of the zones.

In a first embodiment of the device suitable for use in a direct assay,the reagent X may be absent; the reagent Y may be a known amount of theligand under assay, and the reagent Z may be absent. In this embodimentreagent A may be a reactive species immobilised on the surface of theplate being a specific binding partner for the ligand under assay;reagent B may be identical to reagent A; and reagent C may be a reactivespecies immobilised on the surface of the plate being a binding partnernon-specific for the ligand under assay.

In a second embodiment of the device suitable for use in a direct assay,the reagent X may be absent. In such an embodiment, the reagent Y may bea known amount of the ligand under assay together with an amount of aspecific binding partner for the ligand under assay such that a fullysaturated complex exists. In such an embodiment, the reagent Z may beabsent. Thus, zone II carries a known amount of a ligand bound to itsspecific binding partner. Reagent A may be a reactive speciesimmobilised on the surface of the plate being a specific binding partnerfor the ligand under assay; reagent B may be a reactive speciesimmobilised on the surface of the plate being a specific binding partnerfor the reagent Y; and reagent C may be a reactive species immobilisedon the surface of the plate being a binding partner non-specific for theligand under assay.

In a first embodiment of the device suitable for use in a competitiontype assay, the reagent X may be a fluorescently labelled ligandanalogue together with a known amount of a specific binding partner forthe sample ligand under assay. In such an embodiment, the reagent Y maybe a fluorescently labelled ligand analogue together with an amount of aspecific binding partner for the sample ligand under assay such that afully saturated complex exists. In such an embodiment, the reagent Z maybe a known amount of ligand under assay together with an amount of aspecific binding partner for the ligand under assay such that a fullysaturated complex exists. Thus zone I carries a known amount of bothfluorescently labelled ligand analogue and its specific binding partner;zone II carries a known amount of a fluorescently labelled ligandanalogue bound to its specific binding partner; whilst zone III carriesa known amount of the ligand under assay bound to its specific bindingpartner; reagent A may be a reactive species immobilised on the surfaceof the plate, being a specific binding partner for the specific bindingpartner of the sample ligand under assay; and both reagent B and reagentC may be equivalent to reagent A.

In a second embodiment of the device suitable for use in a competitiontype assay, the reagent X may be a fluorescently labelled ligandanalogue; the reagent Y may be the reagent X together with a knownamount of ligand itself; and the reagent Z may be a fluorescentlylabelled ligand analogue together with an amount of a specific bindingpartner for the sample ligand under assay such that a fully saturatedcomplex exists. Thus zone I carries a known amount of a fluorescentlylabelled ligand analogue; zone II carries known amounts of bothfluorescently labelled ligand analogue and the ligand under assay;whilst zone III carries a known amount of a fluorescently labelledligand analogue bound to its specific binding partner. Reagent A may bea reactive species immobilised on the surface of the plate, being aspecific binding partner for the sample ligand under assay; reagent Bmay be equivalent to reagent A; and reagent C may be a reactive speciesimmobilised on the surface of the plate, being a specific bindingpartner for the specific binding partner of the sample ligand underassay.

In a third embodiment of the device suitable for use in a competitiontype assay, the reagent X may be a fluorescently labelled ligandanalogue. In such an embodiment, the reagent Y may be absent and thereagent Z may be equivalent to the reagent X. Thus zone I and zone IIIboth carry a known amount of fluorescently labelled ligand analogue.Reagent A may be a reactive species immobilised on the surface of theplate, being a specific binding partner for the sample ligand underassay; reagent B may be a reactive species immobilised on the surface ofthe plate, being a fluorescently labelled binding partner which may beeither specific or non-specific for the sample ligand under assay; andreagent C may be a reactive species immobilised on the surface of theplate, being a binding partner non-specific for one sample ligand underassay.

In a fourth embodiment of the device suitable for use in a competitiontype assay, the reagent X may be a fluorescently labelled ligandanalogue. In such an embodiment, the reagent Y may be a fluorescentlylabelled ligand analogue together with an amount of a specific bindingpartner for the sample ligand under assay such that a fully saturatedcomplex exists. In such an embodiment, the reagent Z may be the ligandunder assay together with an amount of a specific binding partner forthe ligand under assay such that a fully saturated complex exists. Thuszone I carries a known amount of both fluorescently labelled ligandanalogue and its specific binding partner; zone II carries a knownamount of a fluorescently labelled ligand analogue bound to its specificbinding partner; whilst zone III carries a known amount of the ligandunder assay bound to its specific binding partner. Reagent A may be areactive species immobilised on the surface of the plate, being aspecific binding partner for the sample ligand under assay; and bothreagent B and reagent C may be equivalent to reagent A.

In a fifth embodiment of the device suitable for use in acompetition-type assay, the reagent X may be a fluorescently labelledligand analogue. In such an embodiment, reagent Y may be absent and thereagent Z may be absent or may be equivalent to reagent X. Thus zone Iand optionally zone III both carry a known amount of a fluorescentlylabelled ligand analogue. Reagent A may be a reactive speciesimmobilized on the surface of the plate, being a specific bindingpartner for the sample ligand under assay; reagent B may be the same asreagent A but together with an amount of reagent X such that either afully saturated complex of reagent A with reagent X exists or that saidcomplex forms under the operation of the assay; and reagent C may besame as reagent A but together with an amount of the sample ligand underassay such that either a fully saturated complex of reagent A with thesample ligand under assay exists or that said complex forms under theoperation of the assay and when reagent Z is present, additionallyreagent A together with an amount of a fluorescently labelled ligandanalogue such that either a fully saturated complex of reagent A withthe ligand analogue exists or that said complex forms under theoperation of the assay is present.

In a sixth embodiment of the device suitable for use in a competitiontype assay, the reagent X may be a fluorescently labelled ligandanalogue. In such an embodiment, the reagent Y may be a fluorescentlylabelled ligand analogue together with an amount of a specific bindingpartner for the sample ligand under assay such that a fully saturatedcomplex exists and the ligand under assay together with an amount of aspecific binding partner for the sample ligand under assay such that afully saturated complex exists. In such an embodiment, reagent Z may beequivalent to reagent X. Thus, zone I carries a known amount offluorescently labelled ligand analogue; zone II carries a known amountof a fluorescently labelled ligand analogue bound to its specificbinding partner together with a known amount of the ligand under assaybound to its specific binding partner; whilst zone III carries the samereagent as zone I. Reagent A may be a reactive species immobilised onthe surface of the plate, being a specific binding partner for thesample ligand under assay; reagent B may be a reactive speciesimmobilised on the surface of the plate, being a specific bindingpartner for the specific binding partner of the sample ligand underassay; and reagent C may be a reactive species immobilised on thesurface of the plate, being a binding partner non-specific for thesample ligand under assay.

The measurement regions and some of the calibration regions in theembodiments hereinbefore described are such that the species whichbecomes bound to the immobilised reagent on the measurement orcalibration surface is bound indirectly via one intervening moiety.Further embodiments wherein no intervening moiety is present and thosewherein more than one intervening moiety is present suggest themselvesand are equally within the scope of the present invention. The followingfourteen embodiments relate to the case where no intervening moiety ispresent i.e. the species becomes bound directly to the immobilisedreagent on the measurement or calibration surface.

In a seventh embodiment of the device suitable for use in a competitiontype assay, the reagent X may be a fluorescently labelled ligandanalogue. In such an embodiment, the reagent Y may be the reagent Xtogether with the ligand itself and the reagent Z may be equivalent tothe reagent X or may be absent. Thus, zone I carries a known amount of afluorescently labelled ligand analogue; zone II carries known amounts ofboth fluorescently labelled ligand analogue and the ligand under assay;whilst when reagent Z is present zone III carries the same reagent aszone I. In such an embodiment, reagent A may be a reactive speciesimmobilized on the surface of the plate, being a specific bindingpartner for the sample ligand under assay; reagent B may be equivalentto reagent A; and reagent C may be reagent A together with an amount ofreagent X such that either a fully saturated complex of reagent A withreagent X exists or that said complex forms under the operation of theassay.

In an eighth embodiment of the device suitable for use in acompetition-type assay, the reagent X may be a fluorescently labelledligand analogue. In such an embodiment, the reagent Y may be the same asreagent X but together with the ligand itself and the reagent Z may beequivalent to the reagent X. Thus zone I carries a known amount of afluorescently labelled ligand analogue: zone II carries known amounts ofboth fluorescently labelled ligand analogue and the ligand under assay;whilst zone III carries the same reagent as zone I; reagent A may be areactive species immobilized on the surface of the plate, being aspecific binding partner for the sample ligand under assay; reagent Bmay be equivalent to reagent A; and reagent C may be reagent A togetherwith an amount of the sample ligand under assay such that either a fullysaturated complex of reagent A with the sample ligand under assay existsor that said complex forms under the operation of the assay.

In a ninth embodiment of the device suitable for use in a competitiontype assay, the reagent X may be a fluorescently labelled ligandanalogue. In such an embodiment, the reagent Y may be same as thereagent X but together with the ligand itself and the reagent Z may beequivalent to the reagent X. Thus, zone I carries a known amount of afluorescently labelled ligand analogue; zone II carries known amounts ofboth fluorescently labelled ligand analogue and the ligand under assay;whilst zone III carries the same reagent as zone I; reagent A may be areactive species immobilized on the surface of the plate, being aspecific binding partner for the sample ligand under assay; and reagentB may be equivalent to reagent A; and reagent C may be a reactivespecies immobilized on the surface of the plate, being a specificbinding partner for a ligand other than the sample ligand under assay,optionally together with an amount of the ligand for which it is aspecific binding partner such that a fully saturated complex of saidreactive species with its specific binding partner exists or that saidcomplex forms under the operation of the assay.

In a tenth embodiment of the device suitable for use in a competitiontype assay, the reagent X may be a fluorescently labelled ligandanalogue. In such an embodiment, the reagent Y may be the same asreagent X but together with the ligand itself and the reagent Z may beequivalent to the reagent X. Thus, zone I carries a known amount of afluorescently labelled ligand analogue; zone II carries known amounts ofboth fluorescently labelled ligand analogue and the ligand under assay;whilst zone III carries the same reagent as zone I; reagent A may be areactive species immobilized on the surface of the plate, being aspecific binding partner for the sample ligand under assay; and reagentB may be equivalent to reagent A; and reagent C may be a reactivespecies immobilized on the surface of the plate, being a specificbinding partner for a ligand other than the sample ligand under assay,optionally together with an amount of a fluorescently labelled analogueof a ligand for which it is a specific binding partner such that eithera fully saturated complex of said reactive species with said analogue ofthe ligand that is its specific binding partner exists or that saidcomplex forms under the operation of the assay.

In an eleventh embodiment of the device suitable for use in acompetition-type assay, the reagent X may be a fluorescently labelledligand analogue. In such an embodiment, the reagent Y may be same as thereagent X but together with the ligand itself. In such an embodiment,the reagent Z may be a ligand analogue, said ligand being distinct fromthe sample ligand and not being a specific binding partner for thereactive species for which the sample ligand is a specific bindingpartner. Thus zone I carries a known amount of a fluorescently labelledligand analogue; zone II carries known amounts of both the fluorescentlylabelled ligand analogue and the ligand under assay; whilst zone IIIcarries a known amount of a fluorescently labelled ligand analoguedistinct from the ligand analogue used in zone I ; reagent A may be areactive species immobilized on one surface of the plate, being aspecific binding partner for the sample ligand under assay; and bothreagent B and reagent C may be equivalent to reagent A.

In a twelfth embodiment of the device suitable for use in acompetition-type assay, the reagent X may be a fluorescently labelledligand analogue. In such an embodiment, both the reagent Y and thereagent Z may be equivalent to reagent X. Thus all three zones I, II andIII carry a known amount of a fluorescently labelled ligand analogue;reagent A may be a reactive species immobilized on the surface of theplate, being a specific binding partner for the sample ligand underassay; reagent B may be the same as reagent A but together with anamount of reagent X such that either a fully saturated preformed complexof reagent A with reagent X exists or that said complex forms under theoperation of the assay; and reagent C may be a reactive speciesimmobilized on the surface of the plate, being a specific bindingpartner for a ligand other than the sample ligand under assay,optionally together with an amount of the ligand for which it is aspecific binding partner such that a fully saturated complex of saidreactive species with its specific binding partner exists or that saidcomplex forms under the operation of the assay.

In a thirteenth embodiment of the device suitable for use in acompetition-type assay, the reagent X may be a fluorescently labelledligand analogue. In such an embodiment, the reagent Y may be equivalentto the reagent X. In such an embodiment, the reagent Z may be afluorescently labelled ligand analogue, said ligand being distinct fromthe sample ligand and being a binding partner non-specific for thereactive species for which the sample ligand is a specific bindingpartner. Thus zone I and zone II both carry a known amount of afluorescently labelled ligand analogue; whilst zone III carries a knownamount of a fluorescently labelled ligand analogue distinct from theligand analogue used in zone I; reagent A may be a reactive speciesimmobilized on the surface of the plate, being a specific bindingpartner for the sample ligand under assay; reagent B may be the same asreagent A but together with an amount of reagent X such that either afully saturated complex of reagent A with reagent X exists or that saidcomplex forms under the operation of the assay; and reagent C may be thesame as reagent A optionally together with an amount of the sampleligand under assay such that either a fully saturated complex of reagentA with the sample ligand under assay exists or that said complex formsunder the operation of the assay.

In a fourteenth embodiment of the device suitable for use in acompetition-type assay, the reagent X may be a fluorescently labelledligand analogue. In such an embodiment, the reagent Y may be absent ormay be equivalent to reagent X and the reagent Z may be equivalent toreagent X. Thus zones I and III and optionally zone II carry a knownamount of a fluorescently labelled ligand analogue: reagent A may be areactive species immobilized on the surface of the plate, being aspecific binding partner for the sample ligand under assay; reagent Bmay be a fluorescently labelled reactive species immobilized on thesurface of the plate, optionally being a specific binding partner forthe sample ligand under assay; and reagent C may be reagent A togetherwith an amount of the sample ligand under assay such that either a fullysaturated complex of reagent with the sample ligand under assay existsor that said complex forms under the operation of the assay.

In a fifteenth embodiment of the device suitable for use in acompetition-type assay, the reagent X may be a fluorescently labelledligand analogue. In such an embodiment, the reagent Y may be absent andthe reagent Z may be equivalent to reagent Z. Thus zones I and III carrya known amount of a fluorescently labelled ligand analogue; reagent Amay be a reactive species immobilized on the surface of the plate, beinga specific binding partner for the sample ligand under assay; reagent Bmay be a fluorescently labelled reactive species immobilized on thesurface of the plate, optionally being a specific binding partner forthe sample ligand under assay; and reagent C may be a reactive speciesimmobilized on the surface of the plate, being a specific bindingpartner for a ligand other than the sample ligand under assay,optionally together with an amount of the ligand for which it is aspecific binding partner such that either a fully saturated complex ofsaid reactive species with ligand that is its specific binding partnerexists or that said complex forms under the operation of the assay.

In a sixteenth embodiment of the device suitable for use in acompetition-type assay, the reagent X may be a fluorescently labelledligand analogue. In such an embodiment, the reagent Y may be the same asreagent X but together with the ligand under assay. In such anembodiment, the reagent Z may be the reagent X together with an amountof the ligand under assay, said amount being different to that presentin reagent Y. Thus zone I carries a known amount of a fluorescentlylabelled ligand analogue; both zone II and zone III carry known amountsof both fluorescently labelled ligand analogue and the ligand underassay; reagent A may be a reactive species immobilized on the surface ofthe plate, being a specific binding partner for the sample ligand underassay; and both reagent B and reagent C may be equivalent to reagent A.

In a seventeenth embodiment of the device suitable for use in acompetition type assay, the reagent X may be a fluorescently labelledspecific binding partner for the ligand under assay. In such anembodiment, the reagent Y may be a fluorescently labelled specificbinding partner for the ligand under assay and a fluorescently labelledspecific binding partner for the ligand under assay together with anamount of the ligand under assay such that a fully saturated complexexists. In such an embodiment, the reagent Z may be a fluorescentlylabelled binding partner non-specific for the ligand under assay. Thus,zone I carries a known amount of fluorescently labelled specific bindingpartner for the ligand under assay; zone II carries a known amount offluorescently labelled specific binding partner for the ligand underassay bound to the ligand under assay together with a known amount ofthe unlabelled specific binding partner for the ligand under assay;whilst zone III carries a known amount of a fluorescently labelledbinding partner non-specific for the ligand under assay; reagent A maybe a reactive species immobilised on the surface of the plate, being theligand under assay; and reagent B and reagent C may be identical toreagent A.

In an eighteenth embodiment of the device suitable for use in acompetition type assay, the reagent X may be a fluorescently labelledspecific binding partner for the ligand under assay. In such anembodiment, the reagent Y may be absent. In such an embodiment, thereagent Z may be a fluorescently labelled specific binding partner forthe ligand under assay together with an amount of the ligand under assaysuch that a fully saturated complex exists and a fluorescently labelledligand analogue, said ligand not being the ligand under assay. Thus,zone I carries a known amount of fluorescently labelled specific bindingpartner for the ligand under assay; whilst zone III carries a knownamount of unlabelled specific binding partner for the ligand under assaybound to the ligand under assay together with a fluorescently labelledligand analogue, the ligand being distinct from the ligand under assay;reagent A may be a reactive species immobilised on the surface of theplate, being the ligand under assay; and reagent B may be a reactivespecies immobilised on the surface of the plate, being a fluorescentlylabelled ligand analogue; and reagent C may be a reactive speciesimmobilised on the surface of the plate, being a specific bindingpartner for the ligand under assay.

In a nineteenth embodiment of the device suitable for use in acompetition type assay, the reagent X may be a fluorescently labelledspecific binding partner for the ligand under assay. In such anembodiment, the reagent Y may be a known amount of the ligand underassay together with its specific binding partner such that a fullysaturated complex exists at a specific ratio (e.g. 1:2). In such anembodiment, the reagent Z may be a fluorescently labelled bindingpartner non-specific for the ligand under assay. Thus zone I carries aknown amount of fluorescently labelled specific binding partner for theligand under assay; zone II carries a known amount of fluorescentlylabelled specific binding partner for the ligand under assay bound tothe ligand under assay; whilst zone III carries a known amount of afluorescently labelled binding partner non-specific for the ligand underassay; reagent A may be a reactive species immobilised on the surface ofthe plate, being the ligand under assay; and reagent B may either be areactive species immobilised on the surface of the plate, being aspecific binding partner for the ligand under assay or may be equivalentto reagent A; and reagent C may be equivalent to reagent A.

In a twentieth embodiment of the device suitable for use in acompetition type assay, the reagent X may be a fluorescently labelledspecific binding partner for the ligand under assay. In such anembodiment, the reagent Y may be absent. In such an embodiment, thereagent Z may be a fluorescently labelled binding partner non-specificfor the ligand under assay. Thus zone I carries a known amount offluorescently labelled specific binding partner for the ligand underassay; whilst zone III carries a known amount of a fluorescentlylabelled binding partner non-specific for the ligand under assay;reagent A may be a reactive species immobilised on the surface of theplate, being the ligand under assay; and reagent B may be a reactivespecies immobilised on the surface of the plate, being the ligand underassay, together with a fluorescently labelled specific binding partnerfor the ligand under assay such that either a preformed complex of theligand under assay with its specific binding partner exists or that saidcomplex forms under the operation of the device; and reagent C may beidentical to reagent A.

In the above embodiments of devices suitable for use in competitionassays, where one of the reagents is a fluorescently labelled ligandanalogue this may conveniently be a fluorescently labelled aliquot ofthe ligand under assay.

In a first embodiment of the device suitable for use in a sandwich-typeassay, the reagent X may be a fluorescently labelled specific bindingpartner for the ligand under assay, whilst reagent Y may be a fullysaturated complex of reagent X and a known amount of the ligand itself.In such an embodiment, the reagent Z may be a fluorescently labelledbinding partner non-specific for the ligand under assay. Thus, zone Icarries a known amount of a fluorescently labelled specific bindingpartner; zone II carries a known amount of the fluorescently labelledspecific binding partner bound to the ligand under assay; whilst zoneIII carries a known quantity of a binding partner non-specific for theligand under assay carrying the same fluorescent label as reagent X;reagent A may be a reactive species immobilized on the surface of theplate, being a specific binding partner for the sample ligand underassay; and both reagent A and reagent C may be identical to reagent A.

In a second embodiment of the device suitable for use in a sandwich-typeassay, the reagent X may be a fluorescently labelled specific bindingpartner to the ligand under assay, whilst reagent Y may be a fullysaturated complex of reagent X and a known amount of the ligand itself.In such an embodiment, the reagent Z may be identical to the reagent X.Thus zone I carries a known amount of a fluorescently labelled specificbinding partner to the ligand under assay; zone II carries a knownamount of the fluorescently labelled specific binding partner bound tothe ligand under assay; whilst zone III carries the same reagent as zoneI; reagent A may be a reactive species immobilized on the surface of theplate, being a specific binding partner for the sample ligand underassay; and reagent B may be identical to reagent A; and reagent C may bea reactive species immobilized on a surface of the plate, being aspecific binding partner for a ligand other than the sample ligand underassay, optionally together with an amount of ligand for which it is aspecific binding partner such that either a fully saturated complex ofsaid reactive species with the ligand that is its specific bindingpartner exists or that said complex forms under the operation of theassay.

In a third embodiment of the device suitable for use in a sandwich-typeassay, the reagent X may be a fluorescently labelled specific bindingpartner to the ligand under assay. In such an embodiment, the reagent Ymay be absent. In such an embodiment, the reagent Z may be afluorescently labelled specific binding partner for the ligand underassay together with the ligand under assay in a fully saturated complex.Thus, zone I carries a known amount of a fluorescently labelled specificbinding partner for the ligand under assay; whilst zone III carries aknown amount of the fluorescently labelled specific binding partnerbound to the ligand under assay; reagent A may be a reactive speciesimmobilised on the surface of the plate, being a specific bindingpartner for the ligand under assay; reagent B may be a fluorescentlylabelled reactive species immobilised on the surface of the plate,optionally a specific binding partner for the sample ligand under assay;and reagent C may be reagent A together with an amount of the ligandunder assay such that a full saturated complex exists.

In a fourth embodiment of the device suitable for use in a sandwich-typeassay, the reagent X may be a fluorescently labelled specific bindingpartner to the ligand under assay. In such an embodiment, the reagent Ymay be absent. In such an embodiment, the reagent Z may be afluorescently labelled binding partner non-specific for the ligand underassay. Thus, zone I carries a known amount of a fluorescently labelledspecific binding partner for the ligand under assay; whilst zone IIIcarries a known quantity of a fluorescently-labelled binding partnernon-specific for the ligand under assay; reagent A may be a reactivespecies immobilised on the surface of the plate, being a specificbinding partner for the ligand under assay; reagent B may be afluorescently labelled reactive species immobilised on the surface ofthe plate, optionally a specific binding partner for the sample ligandunder assay; and reagent C may be identical to reagent A.

In a fifth embodiment of the device suitable for use in a sandwich-typeassay, the reagent X may be a fluorescently labelled specific bindingpartner to the ligand under assay. In such an embodiment, the reagent Ymay be a fluorescently labelled specific binding partner for the ligandunder assay together with an amount of the ligand under assay such thata fully saturated complex exists and a further fluorescently labelledspecific binding partner for the ligand under assay. In such anembodiment, the reagent Z may be a fluorescently labelled bindingpartner non-specific for the ligand under assay. Thus, zone I carries aknown amount of a fluorescently labelled specific binding partner forthe ligand under assay; zone II carries a known amount of afluorescently-labelled specific binding partner bound to the ligandunder assay together with a known amount of the fluorescently labelledspecific binding partner for the ligand under assay; whilst zone IIIcarries a known quantity of a fluorescently-labelled binding partnernon-specific for the ligand under assay; reagent A may be a reactivespecies immobilised on the surface of the plate, being a specificbinding partner for the ligand under assay; and reagent B and reagent Cmay both be identical to reagent A.

In a sixth embodiment of the device suitable for use in a sandwich-typeassay, the reagent X may be a fluorescently labelled specific bindingpartner to the ligand under assay. In such an embodiment, the reagent Ymay be a fluorescently labelled specific binding partner for the ligandunder assay together with an amount of the ligand under assay in a fullysaturated complex and a further specific binding partner for the ligandunder assay together with an amount of the ligand under assay in a fullysaturated complex. In such an embodiment, the reagent Z may be afluorescently labelled binding partner non-specific for the ligand underassay. Thus, zone I carries a known amount of a fluorescently labelledspecific binding partner for the ligand under assay; zone II carries aknown amount of fluorescently-labelled specific binding partner bound tothe ligand under assay together with a known amount of unlabelledspecific binding partner bound to the ligand under assay; whilst zoneIII carries a known quantity of a fluorescently-labelled binding partnernon-specific for the ligand under assay; reagent A may be a reactivespecies immobilised on the surface of the plane, being a specificbinding partner for the ligand under assay; and reagent B and reagent Cmay both be identical to reagent A.

In a seventh embodiment of the device suitable for use in asandwich-type assay, the reagent X may be a fluorescently labelledspecific binding partner to the ligand under assay. In such anembodiment, the reagent Y may be absent. In such an embodiment, thereagent Z may be a fluorescently labelled binding partner non-specificfor the ligand under assay. Thus, zone I carries a known amount of afluorescently labelled specific binding partner for the ligand underassay; whilst zone III carries a known quantity of afluorescently-labelled binding partner non-specific for the ligand underassay; reagent A may be a reactive species immobilised on the surface ofthe plate, being a specific binding partner for the ligand under assay;and reagent B may be a reactive species immobilised on the surface ofthe plate being a specific binding partner for the ligand under assaytogether with an amount of the ligand under assay and an amount of afluorescently labelled specific binding partner for the ligand underassay such that a fully saturated complex exists or that said complexforms under the operation of the assay; and reagent C may be identicalto reagent A.

The reasons for using the reagents X, Y, Z, A, B and C described for thevarious embodiments will be discussed later.

In the embodiments of the device hereinbefore described, in the casewhere the ligand under assay is unstable in solution or is rare,expensive or difficult to prepare in a sufficiently pure and/orquantifiable form, in the calibration region(s), wherein the ligandunder assay is used as a calibration reagent, this ligand may bereplaced by a calibrator as described in EP-A-343932.

Capillary fill devices according to the invention may be manufactured bymethods broadly similar no those described in EP-A-171148.

Thus, according to the present invention we also provide a method ofmanufacturing specifically-reactive sample-collecting and testingdevices as described hereinbefore comprising the steps of

(a) forming an array of patches of suitable reagents, carried by zonesI, II and III as described hereinbefore on the surface of a sheetmaterial which is to provide part of a multiplicity of the devices,

(b) forming an array of patches of suitable reagents, carried by zonesIV, V and VI as described hereinbefore on the surface of an additionalstructure, involving, where appropriate the immobilisation ofspecifically reactive species as described hereinbefore, said additionalstructure together with the said sheet material providing for each ofthe multiplicity of devices a cavity for collecting and retaining avolume of sample liquid in contact with the said layers of suitablereagents, the cavity preferably being of capillary dimension, and

(c) separating the sheet material into portions each providing one or aplurality of the sample-collecting and testing devices.

In this process, the zones of reagents contained on the second plate maybe continuous if the reagents contained in the zones are of an identicalnature. Alternatively, the zones of reagents contained on the secondplate, like the zones of reagents contained on the first plate, may bedivided into a pattern of discrete portions, for example as atwo-dimensional array of patches. When such patches are formed, they canbe made, for example, by firstly forming a continuous layer and thenremoving portions thereof to leave the desired pattern of identicalreagent patches. Alternatively, the desired pattern of patches may beapplied directly (for example by screen-printing), such a techniquebeing most applicable to embodiments where; for each of theaforementioned plates, the reagents contained in the zones on said plateare not identical in nature or else are very expensive and their usagehas to be kept to a minimum.

The immobilisation of a specifically reactive species onto the surfaceof the cavity may be carried out directly or indirectly. For example,when the specifically reactive species is an antibody, indirectimmobilisation may be effected by means of an antispecies antibody whichis itself bound to the said surface. Alternatively, immobilisation maybe effected by conjugating an antibody with biotin and complexing withavidin pre-immobilised on the said surface; or vice versa. A furtherexample of indirect immobilisation involves conjugating fluoresceinisothiocyanate (FITC) to the specific binding partner for the speciesunder assay and immobilising anti-FITC antibody onto said surface.Direct immobilisation may be effected by activating the said surface bytreatment with a suitable reagent (e.g. a silanisation reagent such asaminopropyltrimethoxy-silane) to which the antibody can be covalentlycoupled using an appropriate cross-linking reagent (e.g. glutaraldehydeor glycolaldehyde). Alternative techniques well-known to the man skilledin the art may be used for immobilization of the said coating. Haptensand antigens may be immobilised directly onto the surface of the cavityby using appropriate immobilisation chemistry. Alternatively, thesehaptens and antigens may be conjugated to a protein e.g. poly-L-lysineand then immobilised via the protein onto the cavity surface using knownmethods.

For a better understanding of the present invention, reference is madeto the accompanying drawings.

The following description will be made with specific reference to FCFDspossessing one auxiliary calibration surface but it will be appreciatedthat other devices of different design or FCFDs or other devicespossessing different numbers of auxiliary calibration surfaces could besimilarly constructed.

Referring to FIG. 1, the device depicted comprises an upper plate 2fashioned of transparent material (e.g. of plastic material, quartz,silica or glass) carrying on its external face an opaque coating 8, anda lower plate 4 fashioned of transparent material, both plates beingaround 1 mm thick and fixed together in substantially parallelrelationship, less than 1 mm apart by means of bonding tracks 38 (seeFIG. 2) of suitable adhesive. In the embodiment shown, the cell cavity 6so formed is open to the surroundings at both ends, so that when liquidsample is drawn into one opening of the cavity by means of capillarity,air may escape through the other opening. In the embodiment shown, thetwo plates are offset, although this is not a necessary feature.

Carried on the inner surface of the upper plate 2 are three patches ofreagents appropriate to the test being carried out, being carried byzone I (12), zone II (14) and zone III (16) as defined hereinbefore.These reagents are contained within the device in a soluble releasableform (reagents X, Y and Z respectively)

Carried on the inner surface of the lower plate 4 are three patches ofreagent appropriate to the test being carried out, being carried by zoneIV (9), zone V and zone VI (11) as defined hereinbefore, said zones 9,10 and 11 being directly below the zones 12, 14 and 16 respectively onone plate 2. In the case of an immunoassay, the zones 9, 10 and 11 willeach carry for example, a relevant immobilised antibody or antigen orhapten. These are reagents A, B and C.

The operation in use of several embodiments of the device shown in FIG.1 will now be described. Although the following descriptions relate tothe use of a device in a labelled-antigen format competition-typeimmunoassay, it should be understood that devices according to theinvention are also suitable for use in labelled-antibody formatimmunoassays (both competition-type and sandwich-type) and in othertypes of assay (direct, sandwich-type or competition-type) or in othertypes of chemical or biochemical tests.

The sample liquid passes into the device in the direction of the arrowshown in FIG. 1. A short time after the cavity 6 fills with sampleliquid, the matches 12, 14, 16 of material dissolve, releasing therespective reagents into the liquid.

The success of the method of assay of the present invention depends onthe spatial separation (i.e. non-mixing) of the reagents released intothe sample solution from the patches 12, 14, 16. As mentionedhereinbefore, the patches 12, 14, 16 may be carried on the upper plate 2by means of suitable dissoluble material(s). Suitable dissolublematerials include humectant coatings, e.g. sucrose- or sorbitol-based.In the embodiment shown in FIG. 1, the patches 12, 14, 16 are separatedfrom each other. The length of the plates 2, 4 is about 15 mm, thesmallest dimension of the cavity 6 is less than 1 mm (typically about0.1 mm) and one lateral separations 18, 20 between the patches aretypically 2-3 mm. The arrangement of the device is such that when filledwith sample liquid, lateral mixing of reagents is very slow (typically 2or 3 hours), whereas vertical mixing across the narrow capillary gap israpid (several seconds only). Thus, mixing of reagents from the threepatches is not a problem subsequent to the device being filled, sincemost tests (including immunoassays) reach equilibrium in less than 2hours. The possibility of lateral mixing occurring is greatest duringthe filling of the device, when "washdown" of reagents may occur in thedirection of flow of the sample liquid into the device. A furtheroptional precaution against such washdown occurring is to coat thepatches 12, 14, 16 with a thin layer of a material which provides somedelayed release of the reagents within the patches. Suitable materialsfor coating the patches include, for example, polyvinyl alcohol (PVA). Asuitable PVA coating would take typically 2-10 seconds to dissolve afterinitial contact of a sample liquid. In an alternative embodiment to thatshown in FIG. 1, the patches 12, 14 and 16, and thereby thecorresponding patches 9, 10 and 11, may be abutted. There will, in thiscase, be lateral mixing at the interface of the reagent patches.However, the portion of each of the zones which is subsequently selectedfor the purpose of measurement, as described hereinafter, is chosen sothat no mixing will occur between the said portions of adjacent zones.

In one embodiment of the device of the type shown in FIG. 1 which is setup for a competition-type immunoassay for an antigen (which embodimentcorresponds to the first competition-assay embodiment of the devicehereinbefore described), patch 12 may contain a fluorescently labelledantigen analogue together with an amount of a specific antibody to theantigen under assay. Patch 9 would then comprise an immobilised specificbinding partner being a specific antibody to the specific antibody tothe antigen under assay. Thus, upon introduction of the sample liquid,the batch 12 dissolves, releasing antigen analogue and specific antibodyto the antigen under assay into the sample liquid. These reagentsreleased from patch 12 should preferably remain substantially within theregion indicted in FIG. 1 by the label "T". In general, this will be thecase when lateral diffusion is slow. Antigen introduced in the sampleliquid competes with antigen analogue for epitopic binding sites on thespecific antibody to the antigen which, either before or after suchcompetition occurs, becomes bound to the epitopic binding sites on thelayer of specific antibody contained in patch 9. The amount offluorescent material which becomes indirectly bound to the immobilisedspecific antibody in batch 9 will therefore be a function of theconcentration of antigen in the sample liquid. Conventionalcompetition-type optical immunoassays involve this type of competitiveequilibrium. Thus region T acts as the "measurement region". When patch14 dissolves, a known quantity of fluorescently labelled antigenanalogue in a fully saturated complex with its specific antibody isreleased into the sample liquid which is present in the region R asshown in FIG. 1. Thus, by comparison with region T, the antigenanalogue:specific antibody complex becomes bound to the immobilisedspecific antibody contained in patch 10 (which will be identical to thatin patch 9). Thus, initially, a maximum amount of fluorescent materialis indirectly bound to the immobilised antibody in patch 10. Thus, thecalibration region R acts as a "high signal calibration region". After asignificant time period e.g. 1 to 2 hours, the antigen analogue bound tothe immobilised antibody will compete with the antigen from the sampleliquid. This competition will result in a slow decrease in the amount offluorescent material indirectly bound to the immobilised antibody inpatch 11, until the competition has reached equilibrium. When patch 16dissolves, a known quantity of the antigen under assay in a fullysaturated complex with its specific antibody is released into the sampleliquid present in region S as shown in FIG. 1. Thus, in an analogousmanner to region R, the antigen:specific antibody complex becomes boundto the immobilised specific antibody contained in patch 11 (which willbe identical to that in patches 9 and 10). Thus, initially andsubsequently, to fluorescent material becomes indirectly bound to theimmobilised antibody in patch 11. Thus the calibration region S acts asa "zero signal calibration region".

For this first embodiment, the three regions T, R and S and the reagentscontained therein are illustrated diagrammatically in FIG. 3a.

The subsequent descriptions of examples of the device according to thepresent invention are set out in terms of the three regions T, R and Sas defined above. One may use any of the regions T described hereintogether with one of the regions R described herein (in which if abinding reaction occurs at the surface 4 in this region R in isanalogous to that which occurs at the surface 4 in the region T) andoptionally one or more further regions selected from any of regions Rand S described herein.

In a second example of the device for a competition-type assay, thespecific antibody to the antigen under assay may be contained withinpatch 9. In a third example, the specific antibody to the antigen underassay may be prebound to the immobilised antibody contained in patch 9.In a fourth example, the specific antibody to the antigen under assaymay itself be immobilised on the surface containing patch 9. In each ofthese three examples, the resulting competition will be analogous tothat in the measurement region described above, and in these examplesare thereby described further measurement regions.

The region T for these three examples are illustrated diagrammaticallyin FIGS. 3b, 3c and 3d respectively.

In a fifth example the device for a competition-type assay, when patch14 dissolves, a known quantity of fluorescently-labelled antigenanalogue together with a known quantity of the antigen under assay arereleased into the sample liquid which is present in region R as shown inFIG. 1. In general, the quantities of antigen analogue in patches 12 and14 will be the same, although this is not a necessary condition forsuccessful operation of the device. Thus, by comparison with region T,antigen analogue competes with an augmented amount of antigen, forexample sample antigen and antigen already in the device, for bindingsites on the specific antibody which is contained in patch 10. Thisspecific antibody is a specific antibody to the antigen under assay andis present in a saturated complex with an immobilised specific antibodycontained in patch 10. The amount of fluorescent material which becomesindirectly bound to the immobilised specific antibody in patch 10 willtherefore be a function of the concentration of the total amount ofantigen in the region R of the sample liquid. Thus calibration region Racts as a "positive calibration region". In a sixth example, thespecific antibody to the antigen under assay may itself be immobilisedonto the region 10. This example results in an analogous positivecalibration region. In a seventh example, the specific antibody to theantigen under assay may be present in a fully saturated complex withboth the antigen analogue and the antigen 14, the immobilised specificantibody contained in patch 10 being a specific antibody to the specificantibody to the antigen under assay. This example results in a highsignal calibration region.

The region R for these three examples is illustrated diagrammatically inFIGS. 3e, 3f and 3g respectively.

In an eighth example of the device for a competition-type assay, patch14 may contain to reagent. The specific antibody to the antigen underassay is contained within patch 10 together with an equivalent amount offluorescently labelled antigen analogue (the same reagent as in patch12) and together with an equivalent amount of an immobilised specificantibody which is a specific antibody to the specific antibody to theantigen under assay. In a ninth example, the specific antibody to theantigen under assay may be prebound in a complex to the immobilisedspecific antibody in patch 10. In a tenth example, the antigen analogueand specific antibody to the antigen under assay may both be prebound ina complex to the immobilised specific antibody in patch 10. In aneleventh example, the specific antibody to the antigen under assay mayitself be the immobilised antibody in patch 10 together with the antigenanalogue preferably in a fully saturated complex. In each of these fourexamples, initially a maximum amount of fluorescent material becomesbound to the immobilised antibody in patch 10. Thus in these examples,region R acts as a high signal calibration region. After a significanttime period e.g. 1 to 2 hours, the antigen analogue bound to theimmobilised antibody will compete with the antigen from the sampleliquid. This competition will result in a slow decrease in the amount offluorescent material indirectly bound to the immobilised antibody inpatch 11, until the competition has reached equilibrium.

The region R for these four examples is illustrated diagrammatically inFIGS. 3h, 3i, 3j and 3k respectively.

In a twelfth example of the device for a competition-type assay, whenpatch 16 dissolves fluorescently-labelled antigen analogue is releasedinto the sample liquid present in region S as shown in FIG. 1. Thespecific antibody to the antigen under assay is contained within patch11 together with an equivalent amount of the antigen under assay andtogether with an equivalent amount of an immobilised specific antibodywhich is a specific antibody to the specific antibody to the antigenunder assay. In a thirteenth example, the specific antibody to theantigen under assay may be prebound in a complex to the immobilisedspecific antibody in patch 10. In a fourteenth example, the antigenunder assay and specific antibody to the antigen under assay may both beprebound in a complex to the immobilised specific antibody in patch 10.In a fifteenth example, the specific antibody to the antigen under assaymay itself be the immobilised antibody in patch 10 together with theantigen under assay preferably in a preformed complex. In each of thesefour examples, initially to fluorescent material will become bound tothe immobilised antibody in patch 11. Thus in these four examples,region S acts as a zero signal calibration region. After a significanttime period, e.g. 1 to 2 hours, the antigen bound to the immobilisedantibody will become displaced by the fluorescent antigen analoguereleased from patch 16 resulting in a competition between the antigenand the antigen analogue. This competition process can be followed bythe amount of fluorescent material that becomes bound to the immobilisedspecific antibody in patch 11.

The region S for these four examples is illustrated diagrammatically inFIGS. 3l, 3m, 3n and 3p respectively.

In a sixteenth and seventeenth example of the device for acompetition-type assay, when patch 14 dissolves, a fluorescentlylabelled specific antibody, being either a specific antibody for theantigen under assay or being an antibody non-specific for the antigenunder assay, is released into the sample liquid present in region R asshown in FIG. 1. The immobilised antibody which is contained withinpatch 10 will be a specific antibody to the fluorescently labelledantibody contained in patch 14. Thus a maximum amount fluorescentmaterial becomes bound to the immobilised antibody in patch 10. Thusregion R acts as a high signal calibration region.

The region R for these examples are illustrated diagrammatically inFIGS. 3q and 3r respectively.

In an eighteenth example of the device suitable for a competition-typeassay, to reagent is contained in patch 14. The immobilised antibodywhich is contained within patch 10 will be a fluorescently-labelledspecific antibody either for the antigen under assay or for an antigennot being the antigen under assay. In either case, no furtherfluorescent material will become bound to the immobilised antibody inpatch 10. The presence of the fluorescent label on the immobilisedantibody will mean that region R will act as a high signal calibrationregion.

The region R for this embodiment is illustrated diagrammatically in FIG.3s.

In a nineteenth example of the device suitable for a competition-typeassay, when patch 16 dissolves, fluorescently labelled antigen analogue(the same reagent as in patch 12) is released into the sample liquidpresent in region S as shown in FIG. 1. In general, the quantity ofantigen analogue in patch 16 will be the same as the quantity in patch12, although this is not a necessary condition for successful operationof the device. The immobilised antibody which is contained within patch11 will be a binding partner non-specific for the antigen in the sampleliquid. Thus, to fluorescent material will become bound to theimmobilised antibody in patch 11. Thus, region S acts as a zero signalcalibration region.

The region S for this embodiment is illustrated diagrammatically in FIG.3t.

In a twentieth example of the device for a competition-type assay, whenpatch 16 dissolves, a fluorescently labelled antigen analogue, theantigen being distinct from the antigen under assay, is released intothe sample liquid present in region S as shown in FIG. 1. The antibodywhich is contained within patch 11 will be a specific binding partnerfor the antigen under assay. Thus, to fluorescent material will becometo the immobilised antibody in patch 11. Thus, region S acts as a zerosignal calibration region.

The region S for this embodiment is illustrated diagrammatically in FIG.3u.

In a twenty-first example of the device for a competition-type assay,when patch 14 dissolves, fluorescently labelled antigen analogue (thesame reagent as in patch 12) is released into the sample liquid presentin region R as shown in FIG. 1. The antibody which is contained withinpatch 10 will be a binding partner non-specific for the antigen in thesample liquid together with an amount of a fluorescently labelledanalogue of the antigen which is a specific binding partner for theantibody in patch 10 in a complex, which has preferably been preformedin the patch 10. Thus, a maximum amount of fluorescent material becomesbound to the immobilised antibody in patch 10. Thus, region R acts as ahigh signal calibration region.

The region R for this embodiment is illustrated diagrammatically in FIG.3v.

Further examples of embodiments of the device for a competition-typeassay are illustrated in FIGS. 4a to 4t. FIGS. 4a to 4c illustrateexamples of the measurement region T. FIGS. 4d and 4e illustrateexamples of a positive calibration region R. FIGS. 4f to 4m illustrateexamples of a high signal calibration region R. FIGS. 4n to 4rillustrate examples of a zero signal calibration region S. With theexception of FIGS. 4g, 4h, and 4r, these examples include the uselabelled-antibody. FIGS. 4a, 4e, 4f, 4h, 4k and 4l illustrate the use ofa species such as, for example, poly-L-lysine, bovine serum albumin orkeyhole limpet haemocyanin, to facilitate immobilisation of the antigenin one relevant patch. FIG. 4b illustrates an alternative use of aspecies such as, for example, avidin to facilitate the antigen-antibodybinding reaction.

For the direct assay embodiments of the device hereinbefore described,the example of the measurement region T is illustrated in FIG. 5n.Examples of a positive calibration region R are illustrated in FIGS. 5pand 5q. Examples of a high signal calibration region R are illustratedIn FIGS. 5r to 5t. The calibration region S is illustrated in FIG. 5u,being a zero signal calibration region.

For the sandwich assay embodiments of the device hereinbefore described,an example of the measurement region T is illustrated in FIG. 5a. Anexample of a positive calibration region R is illustrated in FIG. 5b.Six examples of a high signal calibration region R are illustrated inFIGS. 5c to 5h. Two examples of a zero signal calibration region S areillustrated in FIGS. 5i and 5j.

Certain examples of calibration regions described for use in acompetition-type assay may find use in a sandwich assay embodiment ofthe device and vice versa. FIGS. 5c and 5g illustrate two such examplesused in both competition-type and sandwich-type assays.

In all of the examples hereinbefore described, the same fluorescentspecies is used as a fluorescent label on those reagents stated to belabelled.

In the embodiment of the device shown in FIG. 1, the patch 12 (beingzone I as defined hereinbefore) is the closest of the three patches onplate 2 to the end of the device where introduction of the sample liquidoccurs, whilst the patch 16 (being zone III) is the furthest of thethree patches on plate 2 from said end of the device. In alternativeembodiments of the device, the patches 12, 14 and 16 and thereby thecorresponding patches 9, 10 and 11 may be arranged in any order from theend of the device where introduction of the sample liquid occurs.

In the various embodiments of the device according to the invention asdefined hereinbefore, one pair of zones provides the measurement regionwhereas the other two pairs of zones provide calibration regions, suchcalibration regions being selected from a positive calibration region, azero signal calibration region or a high-signal calibration region.

In several of the embodiments of the device as defined hereinbefore, inorder to give the desired signal, consideration must be given to thekinetic characteristics of the various binding reactions involved. Thereagents are chosen and the signals from the various regions read at theappropriate time to achieve the desired signal. For certain formats, itis important to ensure that the binding of the intended species occursand to dissociation occurs in any complex initially formed prior to thereading of the signal.

Further embodiments of the device with only one calibration region orwith three or more calibration regions suggest themselves and areincluded within the scope of the present invention, the calibrationregions being preferably selected from those described hereinbefore orthose described hereinafter in the Examples.

Assay measurements are obtained by illuminating in turn with light of anappropriate frequency or range of frequencies the portions of theimmobilised layer (or a part only of said portion) which lies in regionT, region R and region S. This light leads to excitation of fluorophoreswithin the region of illumination. These fluorophores then fluoresce andemit light some or which passes into the second plate 4 and is guided bysaid plate to emerge from the smooth edge 22 with characteristics asdescribed in EP-A-171148, which light may then be filtered and analyzedas desired.

Sequential illumination of the different zones 9, 10 and 11 may beeffected by a shuttering mechanism in the illumination optics, detailsof which will be apparent to one skilled in the art. The optical signalsarising from fluorescent species in each zone will all emerge in turnfrom the optical edge 22 and be detected by the same optical detectorbefore being processed in a desired manner. Alternatively, illuminationof the different regions of the immobilised layer 10 may be effected byuse of a number of identical light sources. Alternatively, it ispossible to use a single light source and index the device past thelight source thereby sequentially illuminating the different regions ofthe immobilised layer 10.

Although the preceding discussion is made with particular reference tofluorescent labels, it will be appreciated that it also applies toreagents conjugated to labels which exhibit other properties (e.g.phosphorescence or luminescence).

FIG. 2 shows a plan view of the lower plate of the device shown inFIG. 1. The patches of material 32, 34, 36 correspond to those labelled12, 14, 16 respectively in FIG. 1. Also shown in FIG. 2 are the bondingtracks 38 which cause mutual adhesion of the upper and lower plates ofthe device. The depth of the capillary gap may be defined byincorporating glass ballotini of appropriate diameter (for example,about 100 microns) in the glue which is used for the bonding tracks 38.

The method of the invention is particularly applicable to assays ofantigens or antibodies, i.e. to immunoassays, and in a preferredembodiment of the invention the ligand is an antigen and the specificbinding partner comprises an antibody to the said antigen. However, theinvention is not to be taken as limited to assays of antibodies orantigens. Examples of ligands which may be assayed by the method of theinvention are given in Table 1 below together with an indication of asuitable specific binding partner in each instance.

                  TABLE 1                                                         ______________________________________                                        Ligand             Specific Binding Partner                                   ______________________________________                                        antigen            specific antibody                                          antibody           antigen                                                    hormone            hormone receptor                                           hormone receptor   hormone                                                    polynucleotide strand                                                                            complementary                                                                 polynucleotide strand                                      avidin             biotin                                                     biotin             avidin                                                     protein A          immunoglobulin                                             immunoglobulin     protein A                                                  enzyme             enzyme cofactor                                                               (substrate) or inhibitor                                   enzyme cofactor    enzyme                                                     (substrate) or inhibitor                                                      lectins            specific carbohydrate                                      specific carbohydrate                                                                            lectins                                                    of lectins                                                                    ______________________________________                                    

The method of the invention has very broad applicability but inparticular may be used to assay: hormones, including peptide hormones(e.g. thyroid stimulating hormone (TSH), luteinizing hormone (LH), humanchorionic gonadotrophin (hCG), follicle stimulating hormone (FSH),insulin and prolactin) or non-peptide hormones (e.g. steroid hormonessuch as cortisol, estradiol, progesterone and testosterone, or thyroidhormones such as thyroxine (T4) and triiodothyronine), proteins (e.g.carcinoembryonic antigen (CEA) and antibodies and alphafetoprotein(AFP)), drugs (e.g. digoxin, drugs of abuse), sugars, toxins, vitamins,viruses such as influenza, para-influenza, adeno-, hepatitis,respiratory and AIDS viruses, virus-like particles or microorganisms.

It will be understood that the term "antibody" used herein includeswithin its scope:

(a) any of the various classes or sub-classes of immunoglobulin, e.g.IgG, IgA, IgM, or IgE derived from any of the animals conventionallyused, e.g. sheep, rabbits, goats or mice,

(b) monoclonal antibodies,

(c) intact molecules or "fragments" of antibodies, monolonal orpolyclonal, the fragments being those which contain the binding regionof the antibody, i.e. fragments devoid of the Fc portion (e.g. Fab,Fab', F(ab')₂), the so-called "half-molecule" fragments obtained byreductive cleavage of the disulphide bonds connecting the heavy chaincomponents in the intact antibody or fragments obtained by syntheticmethods.

The method of preparation of fragments of antibodies is well known inthe art and will not be described herein.

The berm "antigen" as used herein will be understood to include bothpermanently antigenic species (for example, proteins, bacteria,bacterial fragments, cells, cell fragments and viruses) and haptenswhich may be rendered antigenic under suitable conditions.

Examples of fluorophores which may be used in the method of assayaccording to the invention include fluorescein and its derivatives (e.g.fluorescein isothiocyanate (FITC)), rhodamine and its derivatives (e.g.XRITC, TRAP, TRITC), lucifer yellow, 2,4-dinitrofluoro-benzene,phenylisothiocyanate, dansyl chloride, phycobiliproteins (e.g.allophycocyanin and phycoerythrin) and indocyanins.

The present invention further provides apparatus suitable for use in themethod of assay according to the invention as hereinbefore describedwhich comprises a fluorescence capillary fill device according to theinvention as hereinbefore defined; a source of radiation capable ofbeing arranged such that, in use, radiation enters the said device suchthat fluorophores are excited; and means for monitoring the emergingradiation. In a further embodiment, the device can be illuminated via amask, thereby defining the effective volume of the device in which thebinding reaction occurs. The effective volume is the product of thedistance between base and top plates of the device and the area of theillumination zone as defined by the mask 55 in the optical train.

The present invention further provides a kit for performing a method ofassay according to the present invention comprising a device ashereinbefore defined together with appropriate ancillary reagents.

In a quantitative competition assay, it is necessary to have an accuratemeasurement of the concentration of a particular analyte in a sample.Various factors may alter the level of the observed signal in the assayand it is therefore essential to have a sufficient number of definedsignals relating to particular concentrations of analyte to enable astandard assay curve to be constructed. Thus, by using a variety ofcalibration regions wherein the initial binding of fluorophore to thecalibration surface can be pre-determined by using a known amount ofreagents as described in the embodiments hereinbefore, such definedsignals can be achieved which will also compensate for the variousfactors outlined above. In general, known assay techniques employ a 4 or5 point calibration procedure and so for a quantitative assay, it ispreferable to have more than three calibration regions and mostpreferably five or greater.

In a qualitative or semi-quantitative competition assay, it is onlynecessary to determine whether a sample has more or less than a certainconcentration of a particular analyte, this concentration being calledthe `cutoff level` for the particular assay. Therefore, by relating themeasured amount of the analyte in a sample to this `cutoff level` onecan determine whether the sample is `positive` or `negative`. Such a`cutoff level` is generally chosen as the point referring to a 50% levelof binding to the measurement surface of the species giving rise to thesignal although other points may be chosen as the cut-off level.

Similar considerations apply to sandwich assays. In such assays,however, due to the fact that the amount of fluorophore binding to themeasurement surface is directly proportional to the amount of sampleanalyte, a straight-line standard assay graph needs to be constructed.This is easier to achieve than the construction of a standard assay curefor a competion assay. In general, therefore, a quantitative assayrequires only a 3-point calibration procedure; therefore it ispreferable to have 2 calibration regions, more preferably 3 in asandwich-type assay.

In the various embodiments described hereinbefore for either acompetition or sandwich assay, the "high signal calibration regions"have been particularly designed so that an initial maximum amount offluorescent material becomes bound to the surface. However, by alteringthe amounts of the various reagents concerned, different amounts offluorescent material may initially become bound resulting in othernon-zero signals arising from these regions, such amounts being chosento give signals corresponding to the `cutoff level` required. Examplesinclude those illustrated in FIGS. 3g, 3w, 3s, 3v, 3h, 3i, 3j, 3k, 3x,4g, 4h, 4k, 4l, 4t, 5c, 5e, 5f, 5g and 5h.

The "positive calibration regions" as previously described for acompetition-type assay are preferably designed so than the signalrelates to a `cutoff value` corresponding to the inflection point of thestandard assay curve.

The zero signal calibration regions as previously described give asignal corresponding to the background signal for the assay device. Fora competition assay, the regions are designed such that the signalobtained corresponds to the low asymptote of the standard assay curve,whereas for a sandwich assay, the regions are designed such that thesignal corresponds to the lower limit of the standard assay graph.

The following Examples serve to illustrate embodiments of one presentinvention without, however, limiting it.

Examples 1 to 8 illustrate embodiments of the invention in which anantigen-labelled format for a competitive assay of an antigen isdescribed.

EXAMPLE 1 1. PREPARATION OF STARTING MATERIALS 1.1 Fabrication ofAntibody-Coated Waveguides

A sheet of Permabloc glass (Pilkington Glass Ltd., St. Helens, UK)having a thickness of about 1 mm was cleaned with detergent (e.g. TWEEN20 (trademark) Polyoxyethelene sorbitan based detergent) in ultra-purewater with ultrasonic agitation. The surface of the glass was activatedby incubating it in a 2% solution of aminopropyltriethoxysilane in waterat a pH of 3 to 4 for two hours at 75° C. After rinsing in water, theglass sheet was dried at 115° C. for at least four hours. The glass wasthen incubated for 60 minutes in a 2.5% solution of glutaraldehyde in a0.05M phosphate buffer (pH 7), and then wasted thoroughly with distilledwater. The glass was incubated for two to four hours in a 1 percentsolution of a rat anti-mouse monoclonal antibody in phosphate buffer (pH7). The glass sheet was then washed with buffer solution. Unwantedadsorbed protein was removed by soaking with a 6M urea solution in knownmanner. This formed plate 4 of the FCFD test device as illustrated inFIG. 1.

1.2 Preparation of Morphine Conjugated to Fluorescein Isothiocyanate(FITC)

200 mg of FITC (Sigma Chemical Company Ltd., UK) and 5 mg ofmorphine-2-glucuronide were mixed together in 1.4 ml of 0.2M sodiumbicarbonate buffer solution (pH 9.0). The mixture was left for 18 hoursat room temperature, during which conjugation of FITC to the morphineoccurred. The mixture was then purified by gel filtration on SEPHADEXG-50 superfine (beads for gel filtration using low pressure liquidchromotography).

1.3 Microdosing of the Specific Reagents Over Each Discrete ReferenceZone

An opaque coating was screen printed onto a clean sheet of Permablocglass as described in WO-90/14590. The measurement zone (zone I) wasfabricated by microdosing a layer of morphine-FITC conjugate followed bya separate layer of mouse anti-morphine monoclonal antibody in an area3×7 mm onto the glass over zone I. Each layer was allowed to air drybefore a second reagent layer was added on top of it. Since the layersare fabricated in discrete stages, there is to preferential binding ofthe morphine FITC to the anti-morphine monoclonal antibody when thepatient sample is introduced at the time of assay.

In this Example, zone II was fabricated to produce a signal equivalentto the high asymptote of the standard curve from the measurement zone byusing a premix of the mouse anti-morphine monoclonal antibody andmorphine-FITC conjugate accurately microdosed over the zone II area.

Zone III was fabricated to produce a signal equivalent to the lowasymptote of the assay as defined by the measurement standard curve byusing a premix of mouse anti-morphine monoclonal antibody and morphineaccurately microdosed over the zone III area.

This glass sheet containing zones I, II and III forms the plate 2 of theFCFD best device as illustrated in FIG. 1.

1.4 Fabrication of FCFD Test Devices

Test devices such as have been described in EP-A-0171148 were fabricatedby screen printing onto the waveguide resulting from step 1.1 abovebonding tracks of an ultraviolet curing glue (UVS 91, Norland Inc.,U.S.A.) containing glass microspheres of diameter 100 microns (JenconsLtd., UK) in a pattern defining the long edges of the capillary celldevices (see FIG. 2). A sheet of glass as defined in 1.3 above was thenplaced over the waveguide, and a vacuum applied to the laminate. Asresult of the vacuum, the upper sheet of glass was caused to press downonto the glue, the glass microspheres defining a gap of 100 micronsbetween the glass sheets. The laminate was then exposed to anultraviolet light source to cure the glue. Finally, the laminate sheetwas broken into individual test devices as described in EP-A-0171148.

1.5 Preparation of Morphine Standard Solutions

A freeze-dried preparation of morphine-3-glucuronide was obtained fromSigma Chemical Company Ltd. This sample was diluted in pooled humanurine buffered to pH 7.5, to give the range of morphine standardsrequired.

1.6 Apparatus Used in the Measurement of the Morphine Assay

FIG. 6 shows a simple fluorimetry apparatus which was used to makesuitable assay measurements as described in GB8911462.3. Light from azenon flash lamp 51 (Heinmann) is roughly collimated by a lens 52 beforepassing through a filter stack 53 which defines the wavelength rangeused to excite the FITC-labelled antibodies. The filter stack comprisesthree filters: a BG7 Schott glass filter (Ealing Electro Optics UK Ltd.,Watford, UK) a 450-480 nm FITC bandpass interference filter (OptometricsLtd., UK) and a 474 nm shortpass interference filter (Comar InstrumentsLtd., Cambridge, UK). A second lens 54 focused the excitation light ontothe active surface of the test cell 56 through an aperture 55 whichdefines the illuminated area and hence the active volume of the testcell.

Light emitted from the optical edge 63 of the test cell passes throughan aperture 57 which prevents light emitted directly out of the solutionfrom entering the detection optics.

A lens system 58 collects the emitted light and an aperture 59 definesthe angular range over which the emission is measured. This was chosento coincide with angles associated with evanescently coupledfluorescence emission. A Schott OG515 515 nm colloidal glass longpassfiller 60 (Ealing Electro Optics UK Ltd., Watford, UK) filters out anyscattered pump light and a second lens 61 focuses the emission onto aphotomultiplier detector 62 (Hamamatsu P931A, Hakuto UK Ltd).

2. ASSAY PROCEDURE FOR MORPHINE

Eight best CFDs were chosen to produce a standard curve and each wasfilled with a different morphine standard solution. The CFDs were readafter an incubation in a humid environment. Zones V and VI were readafter 3 minutes of incubation, so that the measured signal could beconsidered to represent the high and low asymptote respectively for theassay--(i.e., before significant dissociation had occurred). FIG. 7shows a plot of signal versus time for zone V. It is apparent from thisthat between 0 and 200 seconds the signal is independent of the analyteconcentration, whereas after 200 seconds the labelled reagent begins todissociate from the base plate and competition between the labelledligand analogue and the ligand occurs so that the signal becomesdependent upon the analyte concentration. Hence this zone (and also zoneVI) must be read before 200 seconds have elapsed. Zone IV was read aftera 15 minute incubation (after the assay had reached equilibrium). Fromthis zone, the standard curve was generated (FIG. 8). It will beappreciated that the time before which the reference zone must be read(200 seconds in this particular example) will be dependent on theparticular assay system and reagents used!. FIG. 8 shows that the signalfrom zone IV can be used to derive the analyte concentration in thepatient sample. Zones V and VI produce a signal which can be used to fixthe assay high and low asymptote respectively. At the read time chosen,the signals produced from zones V and VI are independent of analyteconcentration.

Thus the signal from zone IV can be compensated for any change inbackground fluorescence or assay range by using the measured values fromzones V and VI, and comparing them to reference data for zones V and VIobtained during device fabrication.

EXAMPLE 2 1. PREPARATION OF STARTING MATERIALS 1.1 Fabrication ofAntibody-Coated Waveguides

As for example 1, with the exception that zones V and IV were treatedwith a premixed solution of rat anti-mouse and mouse anti-morphineantibodies. Zone VI was not used in this example.

1.2 Preparation of Morphine Conjugated to Fluorescein Isothiocyanate(FITC)

As for example 1.

1.3 Microdosing of the Specific Reagents Over Each Discrete ReferenceZone

As for example 1, except that zone II has a layer of morphine FITCconjugate and unlabelled morphine (either as a premixed solution or asseparate layers) microdosed on to the glass. Zone III is microdosed withmorphine FITC conjugate.

1.4 Fabrication of FCFD Test Devices

As for example 1.

1.5 Preparation of Morphine Standard Solutions

As for example 1.

1.6 Apparatus Used in the Measurement of the Morphine Assay

As for example 1.

2. ASSAY PROCEDURE FOR MORPHINE

The CFDs were filled with a range of morphine standards and read afteran incubation in a humid environment.

Zones IV and V were read when the assay had come to equilibrium--i.e.fifteen minutes. (FIG. 9). Zone IV the assay measurement zone. Zone Vshows an offset when compared to zone IV at low analyte concentrations.This zone is used to define the assay cutoff. Thus when zone IV is xunits larger than zone V, the patient sample is considered to benegative in a competition assay. Furthermore, when the sample from zoneIV is y units less than zone V, the patient sample is considered to bepositive. It is anticipated that zones VI and III would be used tocomplete the calibration regions, by treating the plate carrying zone VIwith rat anti-mouse antibody and the plate carrying zone III with acombined complex of mouse anti-morphine antibody with morphine FITCconjugate.

Zone VI would be read after 3 minutes of incubation so that the measuredsignal could be considered to represent the high asymptote of the assayand is independent of the morphine concentration of the patient sample.Thus zone V would be used to define the cutoff position and zone VI toconfirm that the reagents were working, regardless of patient sampleconcentration.

EXAMPLE 3 1. PREPARATION OF STARTING MATERIALS 1.1 Fabrication ofAntibody-Coated Waveguides

As for example 1, except that zones IV and V were treated with apremixed solution of rat anti-mouse and mouse anti-morphine antibodies.Zone VI was not used in this example.

1.2 Preparation of Morphine Conjugated to Fluorescein Isothiocyanate(FITC)

As for example 1.

1.3 Microdosing of the Specific Reagents Over Each Discrete ReferenceZone

As for example 1 except that zone II has a layer of morphine-FITCconjugate and unlabelled morphine (either as a premixed solution or asseparate layers) microdosed onto the glass and zone I is microdosed withmorphine FITC conjugate. Zone III is not used in this Example.

1.4 Fabrication of FCFD Test Devices

As for example 1.

1.5 Preparation of Morphine Standard Solutions

As for example 1.

1.6 Preparation of Morphine Standard Solutions Using Adulterated Urine

Urine samples were obtained from volunteers known not to be takingmorphine and, after pooling, the samples were treated as follows:

a) The pH of the urine increased to pH 10 by the addition of sodiumhydroxide.

b) The pH of the urine decreased to pH 4.5 by the addition ofhydrochloric acid.

c) The pH of the urine decreased to pH 4.0 by the addition ofhydrochloric acid.

d) The fluorescence of the urine increased by the addition of TRAP togive a final concentration of 1 um/L.

e) The fluorescence of the urine increased by the addition of TRAP togive a final concentration of 6 um/L.

Morphine standard solutions were then made up using these 5 types ofurine as in example 1, giving a range of morphine concentrations aboveand below the assay cut-off.

1.7 Apparatus used in the Measurement of the Morphine Assay

As for example 1.

2. ASSAY PROCEDURE FOR MORPHINE

Assay curves were generated using eight standards in triplicate usingunadulterated urine. Samples made up from the adulterated urines wereassayed and their one signals obtained from the values read off thestandard curve. The signals obtained from the measurement and referencezones were used for each type of sample using the procedure described inexample 2.

FIGS. 10a and 10b show plots of the number of positive results againstdose for various urine types using firstly the standard assay method(FIG. 10a) and then the positive control reference format (10b). Ideallythe step change between positive and negative samples should be abruptbut with the standard assay method, this step change varies with sampletype. Use of the reference zone results in the curves being much moretightly grouped.

EXAMPLE 4 1. PREPARATION OF STARTING MATERIALS 1.1 Fabrication ofAntibody-Coated Waveguides

As for example 1, with the exception that zone V was treated with amouse monoclonal antibody (against hCG) labelled with FITC and zone IVwas treated with a premix of rat antimouse antibody and mouse antimorphine antibody.

1.2 Preparation of Morphine Conjugated to Fluorescein Isothiocyanate(FITC)

As for example 1.

1.3 Microdosing of the Specific Regions Over Each Discreet ReferenceZone

As for example 1 except that the morphine-FITC conjugate was microdosedonto zone I only.

1.4 Fabrication of FCD Test Devices

As for example 1.

1.5 Preparation of Morphine Standard Solutions

As for example 1.

1.6 Apparatus Used in the Measurement of the Morphine Assay

As for example 1.

2. ASSAY PROCEDURE FOR MORPHINE

The CFDs were filled with a range of morphine standards and read afteran incubation period in a humid environment. Both zones IV and V wereread after 15 minutes, although the read time for each zone can beoptimised independently. Zone IV is the measurement zone, and so thesignal is a measure of the analyte concentration. (FIG. 11).

The reagent in zone II is chosen to give a signal from zone V equal tothe high asymptote or the cutoff position. This reference zone correctsfor fluorophore signal strength and patient sample fluorescence, but isnot dependent on assay performance.

One could incorporate a region to provide an assay check using zones IIIand VI. This could be fabricated in a similar way to the region giving asignal equal to the high asymptote in example 2.

EXAMPLE 5 1. PREPARATION OF STARTING MATERIALS 1.1 Fabrication ofAntibody-Coated Waveguides

As for example 1 except that zone IV was treated with a premix of ratanti-mouse and mouse anti-morphine antibodies, while zones V and VI weretreated with rat anti-mouse antibody only.

1.2 Preparation of Morphine Conjugated to Fluorescein Isothiocyanate(FITC)

As for example 1.

1.3 Microdosing of the Specific Reagents Over Each Discrete ReferenceZone

Using the method in example 1, morphine-FITC conjugate was microdosedover zone I, a premix of morphine-FITC conjugate and mouse anti-morphineantibody over zone II and a premix of morphine and mouse anti-morphineantibody over zone III.

1.4 Fabrication of FCFD Test Devices

As for example 1.

1.5 Preparation of Morphine Standard Solutions

As for example 1.

1.6 Apparatus Used in the Measurement of the Morphine Assay

As for example 1.

2. ASSAY PROCEDURE FOR MORPHINE

The CFDs were filled with a range of morphine standards and read afterincubation in a humid environment.

Zone IV was read at 15 minutes, after equilibrium had been reached.Zones V and VI need to be read after shorter incubation times, beforecompetition of the microdosed reagents with the analyte occurs. Thistime is defined by the dissociation of the assay. In this example (FIG.12), zones V and VI were read after 90 seconds.

Under these conditions, zones V and VI produce a signal equivalent tothe high and low asymptote respectively, regardless of the morphineconcentration in the patient sample.

FIG. 13 shows signal plotted against distance from the optical edge ofthe CFD. The read positions for the three zones would typically be at 1mm, 4 mm and 8 mm.

EXAMPLE 6 1. PREPARATION FOR STARTING MATERIALS 1.1 Fabrication ofAntibody-Coated Waveguides

As for example 1, except that all zones were treated with a premix ofrat anti-mouse antibody and mouse anti-morphine antibody.

1.2 Preparation of Morphine Conjugated to Fluorescein Isothiocyanate(FITC)

As for example 1.

1.3 Microdosing of the Specific Reagents Over Each Discrete ReferenceZone

Zone I was fabricated as in example 1, using morphine FITC conjugatemicrodosed onto the plate. The specific reagents were microdosed ontothe antibody-coated waveguide as described in 1.1);

Zone VI was designed to produce a signal equivalent to the highasymptote by microdosing with morphine FITC conjugate, and zone V wasmicrodosed with unlabelled morphine to produce a signal equivalent tothe low asymptote.

1.4 Fabrication of FCFD Test Devices

As for example 1.

1.5 Preparation of Morphine Standard Solutions

As for example 1.

1.6 Apparatus Used in the Measurement of the Morphine Assay

As for example 1.

2. ASSAY PROCEDURE FOR MORPHINE

The CFDs were filled with a range of morphine standards and read afterincubation in a humid environment. Zone IV was read at 15 minutes afterthe assay had come to equilibrium. Zone V and VI were read after 10seconds. The read time was chosen to enable the zones to be measuredbefore the analyte could compete with the microdosed reagents, the timebeing dependent on the dissociation rate of the assay. FIG. 16 shows thedata from zone IV and VI. FIG. 15 shows the data from Zone IV and V.FIG. 14 shows the data from all three zones IV, V and VI.

EXAMPLE 7 1. PREPARATION OF STARTING MATERIALS 1.1 Fabrication ofAntibody-Coated Waveguides

As for example 1 except that zone V was treated with rat anti-mouseantibody and zone IV with a premix of rat anti-mouse and mouseanti-morphine antibody.

1.2 Preparation of Morphine Conjugated to Fluorescein Isothiocyanate(FITC)

As for example 1.

1.3 Microdosing of the Specific Reagents Over Each Discrete ReferenceZone

Using the method outlined in example 1, zone I was microdosed withmorphine-FITC conjugate and zone II with a premix of mouse anti-morphineantibody, morphine and morphine-FITC conjugate.

The combination of morphine and morphine FITC conjugate was used toenable zones II/V to produce a signal equal to the cutoff of the assayrather than the high or low asymptote.

1.4 Fabrication of FCFD Test Devices

As for example 1.

1.5 Preparation of Morphine Standard Solutions

As for example 1.

1.6 Apparatus Used in the Measurement of the Morphine Assay

As for example 1.

2. ASSAY PROCEDURE FOR MORPHINE

The CFDs were filled with a range of morphine standards and read afterincubation in a humid environment. Zone IV, the measurement zone, wasread at 15 minutes after the assay had come to equilibrium. Zone V wasread after 90 seconds. (FIG. 17). This read time was chosen to enablethe signal to be read before the analyte could compete with themicrodosed morphine, the time being dependent on the dissociation rateof the assay.

One could incorporate a region to provide an assay check using zones IIIand VI. zone III would be treated with morphine FITC conjugate and zoneVI with rat anti-mouse antibody only.

The concentration of conjugate in zone III is chosen to give a signalequal to the assay's low asymptote. The conjugate used is the same as inthe measurement zone, but does not bind to the plate carrying zone VI.Thus the signal is equivalent to the assay's background signal.

EXAMPLE 8 1. PREPARATION OF STARTING MATERIALS 1.1 Fabrication ofAntibody-Coated Waveguides

As in example 1 except that a goat anti-mouse antibody premixed withmouse anti-morphine antibody was immobilised onto zone IV of the devicewhilst only goat anti-mouse antibody was immobilised over the rest ofthe surface.

1.2 Preparation of Morphine Conjugated to Rhodamine.

As in example 1 except that rhodamine was substituted for fluorescein.

1.3 Preparation of Antibody Labelled With Rhodamine

Rhodamine was conjugated to a mouse anti-morphine antibody usingestablished techniques.

1.4 Micodosing of Specific Reagents Over Each Reference Zone

As for example 1 except that zone I had only TRAP labelled morphineprinted on it whilst zone II had only TRAP labelled mouse anti-morphineantibody. Zone III was not used in this example.

1.5 Preparation of Morphine Standard Solutions

As for example 1.

1.6 Morphine Samples

Urine samples containing a range of morphine concentrations wereobtained and assayed using a commercially available assay prior to assayin the FCFD.

1.7 Apparatus Used in the Measurement of the Morphine Assay

As for example 1.

2. ASSAY PROCEDURE FOR MORPHINE

Assay curves were generated using eight standards in triplicate usingunadulterated urine. Samples made up from the adulterated urines wereassayed and their the signals obtained from the values read off thestandard curve.

The reagent in zone II is chosen to give a signal equal to the cut offposition. This reference zone corrects for fluorophore signal strengthand patient sample fluorescence. It is also dependent on the assayperformance.

No binding of sample occurs to the reagent in zone VI. Thus the signalfrom this region is equivalent to the assay's background signal.

The following table shows that the use of the reference zone in thisassay improves the overall performance of the assay.

    ______________________________________                                                    Without  With                                                                 Reference Zone                                                                         Reference Zone                                           ______________________________________                                        Number of True                                                                              599        606                                                  negative samples                                                              Number of True                                                                              198        199                                                  positive samples                                                              Number of False                                                                             12         11                                                   negative samples                                                              Number of False                                                                             7          0                                                    positive samples                                                              % correlation 97.7       98.7                                                 overall                                                                       % correlation 94.3       94.5                                                 positive samples                                                              % correlation 98.8       100.0                                                negative samples                                                              ______________________________________                                    

SIGNAL PROCESSING

The previous worked examples have demonstrated various methods formeasuring either the high and low asymptote or the assay's cutoff value.Various methods have then been used to correct the data from themeasurement region by the calibration region data.

These methods can be summarised as either an additive, multiplicative orcombined additive/multiplicative method. All methods rely oncharacterisation of the calibration reasons during manufacture, so thatany difference measured at the time of assay can be used to correct thedata from the measurement region.

The most straightforward method is to directly fix the cutoff valueusing a calibration region. However not all proposed examples are ableto achieve this and so the cutoff may be calculated from the high andlow asymptote.

In FIGS. 3a to 3x, 4a to 4t and 5a to 5u, which illustrate the regionsT, R and S in various embodiments of the device of FIG. 1, the symbolsillustrated denote the following entities:

∘ Antigen under assay

fluorescent label

fluorescently labelled antigen analogue

□ antigen, distinct from antigen under assay or specific antibody toantigen under assay

specific antibody to specific antibody to antigen under assay

or antibody non-specific to the antigen under assay

specific antibody to an antibody non-specific to the antigen under assay

species to facilitate immobilisation or

facilitate antibody-antigen binding specific binding partner to thespecies .

FIG. 6 shows schematically a simple fluorometry apparatus for takingmeasurements from the device of FIG. 1.

FIG. 7 shows a plot of the signal obtained versus the time at which thesignal is measured for zone V of a device used in the assay methoddescribed in Example 1 at two differing concentrations (0 ng/ml and100,000 (100K) ng/ml) of morphine-3-glucuronide in the morphine standardsolution.

FIG. 8 shows a plot of thee signal obtained versus the log.concentration of morphine-3-glucuronide in the morphine standardsolution for zone IV of a device used in the assay method described inExample 1.

FIG. 9 shows a plot of the signal obtained versus the log. concentrationof morphine-3-glucuronide in the morphine standard solution for zones IVand V of a device used in the assay method described in Example 2.

FIGS. 10a and 10b show a plot of the number of positive results obtainedversus the log. concentration of morphine-3-glucuronide in the morphinestandard solution for the sample types described in Example 3 using astandard assay method and an assay method described in Example 3respectively. The following symbols used in FIGS. 10a and 10b denote thesample type used:

□ normal unadulterated urine

⋄ urine at pH 10

Δ urine with a fluorescence level of 1 um/L

+ urine with a fluorescence level of 6 um/L.

FIG. 11 shows a plot of the signal obtained versus the log.concentration of morphine-3-glucuronide in the morphine standardsolution for zones IV and V of a device used in the assay methoddescribed in Example 4.

FIG. 12 shows a plot of the signal versus the log. concentration ofmorphine-3-glucuronide in the morpnine standard solution for zones IV, Vand VI (denoted by the symbols Δ, + and ⋄ respectively) of a device usedin the assay method described in Example 5.

FIG. 13 shows a plot of the signal obtained versus the vernier distancefrom the optical edge of a device used in the assay method described inExample 5.

FIG. 14 shows a plot of the signal obtained versus the log.concentration of morphine-3-glucuronide in the morphine standardsolution for zones IV, V and VI of a device used in the assay methoddescribed in Example 6.

FIG. 15 shows a plot of the signal obtained versus the log.concentration of morphine-3-glucuronide in the morphine standardsolution for zones IV and V of a device used in the assay methoddescribed in Example 6.

FIG. 16 shows a plot of the signal obtained versus the log.concentration of morphine-3-glucuronide in the morphine standardsolution for zones V and VI of a device used in the assay methoddescribed in Example 6.

FIG. 17 shows a plot of the signal obtained versus the log.concentration of morphine-3-glucuronide in the morphine standardsolution for zones IV and V (denoted by the symbols Δ and ⋄respectively) of a device used in the assay method described in Example7.

We claim:
 1. A method of manufacturing a plurality of capillary-filldevices, each device for assay of at least one ligand in a liquid samplecomprising the steps of(a) forming an array of patches for each ligandcomprising at least two mutually separated zones I and II wherein eachzone comprises a releasable layer of a first specific binding reagentselected from the group consisting of the ligand, an analogue of theligand, and a binding partner capable of specifically binding with theligand and the analogue, if present, disposed on a surface of a sheetmaterial which is to provide a part of each of said plurality of thedevices such that each first specific binding reagent does not mix withthe other upon release during performance of the assay, (b) forming asecond array of patches comprising at least two zones IV and Vcorresponding in orientation to the aforementioned zones I and IIrespectively, each of zones IV and V comprising a correspondingimmobilized layer of a second specific binding reagent selected from thegroup consisting of said first specific binding reagent and an auxiliaryspecific binding reagent capable of specifically binding with said firstspecific binding reagent on a surface of an additional structure, saidadditional structure together with the sheet material providing for eachof the plurality of the devices a cavity of capillary dimensions forcollecting and retaining a volume of the liquid sample in contact withthe layers of the releasable reagents, (c) combining the sheet materialand the additional structure to form said plurality of the devices, andseparating the plurality of devices into portions each portion providingsaid each device.
 2. A competitive specific binding method fordetermining a ligand in a liquid sample, comprising:(A) contacting theliquid sample to a capillary-fill device, said device comprising ameasurement zone and at least one spatially distinct calibration zonewherein(1) the measurement zone comprises (a) a known amount of areleasable first ancillary specific binding reagent and (b) ameasurement surface comprising an immobilized measurement specificbinding reagent capable of specifically binding with at least one of theligand and the first ancillary specific binding reagent, wherein thefirst ancillary specific binding reagent is provided either on a surfaceof the measurement zone separate from the measurement surface orprebound to the immobilized measurement specific binding reagent, and iseither (i) labelled ligand analogue or (ii) labelled specific bindingpartner for the ligand or (iii) a combination of the labelled ligandanalogue and specific binding partner for the ligand, (2) thecalibration zone comprises(c) a known amount of a releasable secondancillary specific binding reagent and a calibration surface comprisingan immobilized calibration specific binding reagent having binding sitesidentical in structure to those of the immobilized measurement specificbinding reagent and capable of specifically binding with at least one ofthe ligand and the second ancillary specific binding reagent or (d) acalibration surface comprising a known amount of immobilized label,wherein the second ancillary specific binding reagent is provided eitheron a surface of the calibration zone separate from the calibrationsurface or prebound to the immobilized calibration specific bindingreagent, and is (i) said labelled ligand analogue or (ii) said labelledspecific binding partner for the ligand or (iii) said combination of thelabelled ligand analogue and specific binding partner for the ligand,and (3) the releasable reagents remain in their respective zones withoutmixing with each other in the method following either sequential orsimultaneous contact with the liquid sample (a) to form a labelledmeasurement complex immobilized on the measurement surface in an amountdependent on the amount of the ligand present in the liquid sample and(b) to provide a calibration signal by (c) forming either a labelledcalibration complex immobilized on the calibration surface in an amounteither dependent on or independent of the amount of the ligand presentin the liquid sample or (d) due to said known amount of immobilizedlabel on the calibration surface; and (B) measuring and comparing theamount of label on the measurement and calibration surfaces to determinethe presence or amount of the ligand in the liquid sample.
 3. The methodas claimed in claim 2 wherein the label is a fluorogenic, phosphorogenicor luminogenic label.
 4. The method as claimed in claim 2 wherein themethod is an antigen-antibody immunoassay.
 5. The method as claimed inclaim 2, wherein the capillary-fill device further comprises a spatiallydistinct first auxiliary calibration zone comprising(e) either a knownamount of a releasable third ancillary specific binding reagent and afirst auxiliary calibration surface comprising an immobilized firstauxiliary calibration specific binding reagent having binding sitesidentical in structure to those of the immobilized measurement specificbinding reagent and capable of specifically binding to at least one ofthe ligand and the third ancillary specific binding reagent, or (f) afirst auxiliary calibration surface comprising an immobilized firstauxiliary calibration nonspecific binding reagent, wherein the thirdancillary specific binding reagent is provided either on a surface ofthe first auxiliary calibration zone separate from the first auxiliarycalibration surface or prebound to the immobilized first auxiliarycalibration specific binding reagent, and is (i) said labelled ligandanalogue or (ii) said combination of the labelled ligand analogue andspecific binding partner for the ligand or (iii) an optionally labellednonspecific binding ligand which binds to the immobilized firstauxiliary calibration nonspecific binding reagent, wherein step (A)further comprises (e) forming either a labelled first auxiliarycalibration complex immobilized on the first auxiliary calibrationsurface in an amount either dependent on or independent of the amount ofthe ligand present in the liquid sample, or (f) forming an immobilizednonspecific binding complex in an amount dependent upon nonspecificbinding background, and wherein step (B) further comprises measuring andcomparing the amount of label on the immobilized first auxiliarycalibration surface.
 6. A direct specific binding method for determininga ligand in a liquid sample, comprising:(a) contacting the liquid sampleto a capillary-fill device, said device comprising (1) a measurementzone, (2) at least one spatially distinct calibration zone and (3) atleast one spatially distinct auxiliary calibration zone wherein(1) themeasurement zone comprises (a) a known amount of a releasable firstancillary specific binding reagent and (b) a measurement surfacecomprising an immobilized measurement specific binding reagent capableof specifically binding with at least one of the ligand and the firstancillary specific binding reagent, wherein the first ancillary specificbinding reagent is provided either on a surface of the measurement zoneseparate from the measurement surface or prebound to the immobilizedmeasurement specific binding partner reagent, and is a specific bindingpartner for the ligand, (2) the calibration zone comprises(c) a knownamount of at least one releasable second ancillary specific bindingreagent and a calibration surface comprising an immobilized calibrationspecific binding reagent having binding sites identical in structure tothe those of the immobilized measurement specific binding reagent andcapable of specifically binding with at least one of the ligand and thesecond ancillary specific binding reagent, or (d) a calibration surfacecomprising a known amount of an immobilized calibration complexcomprising the immobilized calibration specific binding reagent andeither the ligand or the second ancillary specific binding reagent,wherein the second ancillary specific binding reagent is provided eitheron a surface of the calibration zone separate from the calibrationsurface or prebound to the immobilized calibration specific bindingreagent, and is (i) said ligand analogue or (ii) said specific bindingpartner for the ligand, and (3) the auxiliary calibration zonecomprises(e) either a known amount of at least one releasable thirdancillary specific binding reagent and a first auxiliary calibrationsurface comprising an immobilized first auxiliary calibration specificbinding reagent having binding sites identical in structure to those ofthe immobilized measurement specific binding reagent and capable ofspecifically binding to a least one of the ligand and the thirdancillary specific binding reagent, or (f) a first auxiliary calibrationsurface comprising an immobilized first auxiliary nonspecific bindingreagent, wherein the third ancillary specific binding reagent isprovided either on a surface of the first auxiliary calibration zoneseparate from the first auxiliary calibration surface or prebound to theimmobilized first auxiliary calibration specific binding reagent, and is(i) said ligand analogue or (ii) said specific binding partner for theligand or (iii) a nonspecific binding ligand which binds to theimmobilized first auxiliary nonspecific binding reagent,(4) thereleasable reagents remain in their respective zones without mixing witheach other in the method following either sequential or simultaneouscontact with the liquid sample (a) to form an unlabelled measurementcomplex immobilized on the measurement surface in an amount dependent onthe amount of the ligand present in the liquid sample, (b) to provide anunlabelled calibration signal by (c) forming either an unlabelledcalibration complex immobilized on the calibration surface in an amounteither dependent on or independent of the amount of the ligand presentin the liquid sample or (d) due to said known amount of an immobilizedcalibration complex comprising the immobilized calibration specificbinding reagent and either the ligand or the second ancillary specificbinding reagent, and to provide an unlabelled auxiliary calibrationsignal by (d) forming either an unlabelled first auxiliary calibrationcomplex immobilized on the first auxiliary calibration surface in anamount either dependent on or independent of the amount of the ligandpresent in the liquid sample or (f) forming an unlabelled nonspecificbinding complex in an amount dependent upon nonspecific bindingbackground, and (B) measuring and comparing a change in property of themeasurement, calibration and auxiliary calibration surfaces due to anychange in mass on the measurement, calibration and auxiliary calibrationsurfaces due to the formation of said unlabelled measurement,calibration and first auxiliary calibration complexes to determine thepresence or amount of the ligand in the liquid sample.
 7. A sandwichspecific binding method for determining a ligand in a liquid sample,comprising:(A) contacting the liquid sample to a capillary-fill device,said device comprising a measurement zone and at least one spatiallydistinct calibration zone wherein:(1) the measurement zone comprises (a)a known amount of a releasable first ancillary specific binding reagentand (b) a measurement surface comprising an immobilized measurementspecific binding reagent capable of specifically binding to at least oneof the ligand and the first ancillary specific binding reagent, whereinthe first ancillary specific binding reagent is provided either on asurface of the measurement zone separate from the measurement surface orprebound to the immobilized measurement specific binding reagent, islabelled or unlabelled, and specifically binds the ligand at an epitopedifferent from an epitope to which the measurement specific bindingreagent or said first ancillary specific binding reagent specificallybinds to, and (2) the calibration zone comprises(c) a known amount of areleasable second ancillary specific binding reagent and a calibrationsurface comprising an immobilized calibration specific binding reagenthaving binding sites identical in structure to those of the immobilizedmeasurement specific binding reagent and which is capable ofspecifically binding with at least one of (i) an unknown amount of theligand, (ii) a known amount of the ligand and (iii) the second ancillaryspecific binding reagent, or (d) a calibration surface comprising aknown amount of immobilized label, wherein the second ancillary specificbinding reagent is provided either on a surface of the calibration zoneseparate from the calibration surface or prebound to the immobilizedcalibration specific binding reagent, and is (i) a labelled orunlabelled specific binding partner for the ligand or (ii) a knownamount of the ligand prebound to a labelled specific binding partner forsaid ligand, and (3) the releasable reagents remain in their respectivezones without mixing with each other in the method following eithersequential or simultaneous contact with the liquid sample (a) to form alabelled measurement complex immobilized on the measurement surface inan amount dependent on the amount of the ligand present in the liquidsample and (b) to provide a calibration signal by (c) forming either alabelled calibration complex immobilized on the calibration surface inan amount either dependent on or independent of the amount of the ligandpresent in the liquid sample or (d) due to said known amount ofimmobilized label on the calibration surface; and (B) measuring andcomparing the amount of label on the measurement and calibrationsurfaces to determine the presence or amount of the ligand in the liquidsample.
 8. The method as claimed in claim 7 wherein the label is afluorogenic, phosphorogenic or luminogenic label.
 9. The method asclaimed in claim 8 wherein the method is an antigen-antibodyimmunoassay.
 10. The method as claimed in claim 7, wherein the method isan antigen-antibody immunoassay.
 11. The method as claimed in claim 7,the capillary-fill device further comprises a spatially distinct firstauxiliary calibration zone comprising(e) either a known amount of areleasable third ancillary specific binding reagent and a firstauxiliary calibration surface comprising an immobilized first auxiliarycalibration specific binding reagent having binding sites identical instructure to those of the immobilized measurement specific bindingreagent and capable of specifically binding to at least one of anunknown amount of the ligand, a known amount of the ligand, and thethird ancillary specific binding reagent, or (f) a first auxiliarycalibration surface comprising an immobilized first auxiliarycalibration nonspecific binding reagent, wherein the third ancillaryspecific binding reagent is provided either on a surface of the firstauxiliary calibration zone separate from the first auxiliary calibrationsurface or prebound to the immobilized first auxiliary calibrationspecific binding reagent, and is (i) a labelled specific binding partnerfor said ligand, (ii) a known amount of the ligand prebound to saidspecific binding partner for said ligand, (iii) a labelled or unlabellednonspecific binding ligand which binds to the immobilized firstauxiliary calibration nonspecific binding reagent, or (iv) a combinationof said nonspecific binding ligand and a labelled or unlabellednonspecific binding reagent therefore, wherein step (A) furthercomprises (e) forming either a labelled first auxiliary calibrationcomplex immobilized on the first auxiliary calibration surface in anamount either dependent on or independent of the amount of the ligandpresent in the liquid sample, or (f) forming an immobilized nonspecificbinding complex in an amount dependent upon nonspecific bindingbackground, andwherein step (B) further comprises measuring andcomparing the amount of label on the immobilized first auxiliarycalibration surface.
 12. A capillary-fill biosensor device suitable foruse in assaying a ligand in a liquid sample, said device comprising (1)a measurement zone, (2) at least one spatially distinct calibration zoneand, optionally, (3) at least one spatially distinct auxiliarycalibration zone wherein(1) the measurement zone comprises (a) a knownamount of a releasable first ancillary specific binding reagent and (b)a measurement surface comprising an immobilized measurement specificbinding reagent capable of specifically binding with at least one of theligand, the first ancillary specific binding reagent, or a first bindingpartner which specifically binds to said ligand and said first ancillaryspecific binding reagent, wherein the first ancillary specific bindingreagent is provided either on a surface of the measurement zone separatefrom the measurement surface or prebound to said immobilized measurementspecific binding partner reagent, (2) the calibration zone comprises(c)a known amount of at least one releasable second ancillary specificbinding reagent and a calibration surface comprising an immobilizedcalibration specific binding reagent having binding sites identical instructure to those of the immobilized measurement specific bindingreagent and capable of specifically binding with at least one of theligand, the second ancillary specific binding partner and a secondbinding partner which specifically binds to said ligand and said secondancillary specific binding reagent, and, optionally, (3) the auxiliarycalibration zone comprises(d) an auxiliary calibration surfacecomprising an immobilized first auxiliary nonspecific binding reagent,wherein the releasable reagents remain in their respective zones withoutmixing with each other in the assay following either sequential orsimultaneous contact with the liquid sample to provide (a) a specificbinding complex immobilized on the measurement surface which iscorrelative of the presence or amount of the ligand in the liquidsample, (b) a calibration specific binding complex immobilized on thecalibration surface as a result of an identical specific bindingreaction as occurs on the measurement surface so as to provide ameasurement correlative of a known amount of said ligand and,optionally, (c) a nonspecific binding complex immobilized on theauxiliary calibration zone to provide a measurement for correctingnonspecific background binding.
 13. The device as claimed in claim 12wherein said measurement, calibration and auxiliary calibration zonesare provided as a transparent capillary comprising an upper portion anda lower portion, wherein the releasable reagents are provided on aninner surface of said upper portion and the measurement, calibration andauxiliary calibration surfaces are provided on an inner surface of saidbottom portion, said transparent capillary being a light-transmissivewaveguide.
 14. The device as claimed in claim 13 further comprising alayer of light-absorbing or opaque material coating an outer surface ofsaid bottom portion of said capillary opposite the measurement,calibration and auxiliary calibration surfaces.
 15. The device asclaimed in claim 13 further comprising said auxiliary calibration zone.16. The device as claimed in claim 12 further comprising a detectionmeans for measuring the specific binding complex immobilized on themeasurement surface, the calibration specific binding compleximmobilized on the calibration surface and, optionally, the nonspecificbinding complex immobilized on the auxiliary calibration zone.
 17. Thedevice as claimed in claim 16 wherein the detection means is afluorescence monitor, the releasable reagents are labeled with afluorogenic label, and wherein the device further comprises a radiationsource for exciting said fluorogenic label.
 18. A specific bindingmethod for determining a ligand in a liquid sample comprising:(a)contacting the liquid sample with the capillary-fill biosensor device asclaimed in claim 12 and (b) measuring and comparing the amount of thespecific binding complex immobilized on the measurement surface, theamount of the calibration specific binding complex immobilized on thecalibration surface as a result of an identical specific bindingreaction as occurs on the measurement surface so as to provide ameasurement correlative of a known amount of said ligand and,optionally, the amount of the nonspecific binding complex immobilized onthe auxiliary calibration zone to correct for nonspecific backgroundbinding in order to determine the presence or amount of the ligand inthe liquid sample.
 19. The method as claimed in claim 18 wherein thereleasable reagents in the capillary-fill biosensor device are labeledwith a fluorogenic, phosphorogenic or luminogenic label.
 20. The methodas claimed in claim 19 wherein the method is an antigen-antibodyimmunoassay.
 21. The method as claimed in claim 18 wherein the method isan antigen-antibody immunoassay.